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Intelligent Design, the best explanation of Origins » Origin of life » Chemistry » Iron Uptake and Homeostasis in Prokaryotic Microorganisms

Iron Uptake and Homeostasis in Prokaryotic Microorganisms

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Iron Uptake and Homeostasis in Cells 1

http://reasonandscience.catsboard.com/t2443-iron-uptake-and-homeostasis-in-prokaryotic-microorganisms

Iron uptake and metabolization in all bacterial species is a major prerequisite for their cell survival due to the importance of this metal in many essential (redox) enzymes. 48

The origin of life required two processes that dominated:
(1) the generation of a proton gradient and
(2) linking this gradient to ATP production in part and in part to uptake of essential chemicals and rejection of others. The generation of a proton gradient required especially appropriate amounts of iron (Fe2+), levels for electron transfer and the ATP production depended on controlling H+, Mg2+ and phosphate in the cytoplasm. 8

Iron serves essential functions in both prokaryotes and eukaryotes, and cells have highly specialized mechanisms for acquiring and handling this metal. 2 Organisms use a variety of transition metals as catalytic centers in proteins, including iron, copper, manganese, and zinc. Iron is well suited to redox reactions due to its capability to act as both an electron donor and acceptor. In eukaryotic cells, iron is a cofactor for a wide variety of metalloproteins involved in energy metabolism, oxygen binding, DNA biosynthesis and repair, synthesis of biopolymers, cofactors, and vitamins, drug metabolism, antioxidant function, and many others. Because iron is so important for survival, organisms utilize several techniques to optimize uptake and storage to ensure maintenance of sufficient levels for cellular requirements. However, the redox properties of iron also make it extremely toxic if cells have excessive amounts. Free iron can catalyze the formation of reactive oxygen species such as the hydroxyl radical, which in turn can damage proteins, lipids, membranes, and DNA. Cells must maintain a delicate balance between iron deficiency and iron overload that involves coordinated control at the transcriptional, post-transcriptional, and post-translational levels to help fine-tune iron utilization and iron trafficking.  4

Question: Had this coordination not have to be fully set up right when cells first became alive?

Iron (Fe) is the most abundant element in the Earth and the fourth most abundant element in the crust. 6 Iron encompasses roughly 5 percent by mass of today's crust. Due to the abundance of iron and its role in biological systems, the transition and mineralogical stages of iron have played a key role in Earth surface systems. Iron played a larger role in the geological past in marine geochemistry, as evidenced by the deposits of Precambrian iron-rich sediments. The redox transformation of Fe(II) to Fe(III), or vice versa, plays a big role in a number of biological and element cycling processes. The reduction of Fe(III) is seen to oxidize sulfur (from H2S to SO4-2), which is a central process in marine sediments. Many of the first metalloproteins consisted of iron-sulfur complexes formed during photosynthesis. Iron is the main redox metal in biological systems. In proteins, it is found in a variety of sites and cofactors, including, for instance, haem groups, Fe–O–Fe sites, and iron-sulfur clusters. The prevalence of iron is apparently due to the large availability of Fe(II) in the initial emergence of living organisms, before the rise photosynthesis and of atmospheric oxygen levels which resulted in the precipitation of iron in the environment as Fe(OH)3.

Iron can be taken up selectively as ferredoxins, Fe-O-Fe (hemerythrin and ribonucleotide reductase), Fe (many oxidases), apart from iron porphyrin. Variation in the related proteins with any one of these chemical forms of iron has produced a wide range of enzymes. It is important to understand that all these arrangements are modified to function both in the sense of reactivity and its positioning of the protein in the cell. In many cases the iron can have many redox and spin states and, it can be held in many stereochemistries.

Eukaryotic cells contain hundreds of proteins that require iron cofactors for activity. 3 These iron enzymes are located in essentially every subcellular compartment; thus, iron cofactors must travel to every compartment in the cell. Iron cofactors exist in three basic forms: Heme, iron-sulfur clusters, and simple iron ions (also called non-heme iron). Emerging evidence suggests that specific systems exist for the distribution of iron cofactors within the cell. These systems include membrane transporters, protein chaperones, specialized carriers, and small molecules.

One of the most important concepts to emerge from the field of eukaryotic cell biology is that the contents of the cell are highly organized and the movement of organelles and macromolecules within the cell does not occur through simple diffusion. Movement is highly regulated through packaging and assembly of complexes and through directed transport via components of the cytoskeleton. Cells express hundreds, perhaps thousands of proteins that require bound metal cofactors for activity and stability.  Iron and zinc are the most abundant metals in cells, followed by copper and manganese, and, to a lesser extent, cobalt, nickel, and molybdenum. Thus, it is not surprising to discover the existence of intracellular systems for the distribution of metals and metal cofactors. Why do cells need these metal delivery systems? It is because the cell faces several obstacles in achieving the two major goals of metal cofactor delivery, which are the incorporation of the native cofactor and exclusion of non-native cofactors.

The sulfur, oxygen, and nitrogen ligands that coordinate metals in enzymes will frequently bind non-native cofactors with affinities equal to or greater than those of the native cofactor. A second obstacle is that redox-active metals, such as iron, copper, and manganese can engage in Fenton-type chemistry in the presence of oxygen and produce potentially damaging reactive oxygen species. Thus, cells must tightly regulate the uptake, storage, and distribution of metal ion species. A third obstacle is that zinc and copper ions exhibit the highest affinity for transition metal-binding sites and would occupy iron and manganese sites if the metals were present in freely exchangeable pools of similar concentrations.

Consequently, cells maintain pools of zinc and copper at exceedingly low levels. This is accomplished through tightly regulated uptake and efflux and through the expression of metal-binding proteins, such as  metallothioneins, that effectively sequester pools of zinc and copper. Iron, however, appears to be managed differently.

Iron Bioavailability 4
Although iron is one of the most abundant elements on Earth, the environment is usually oxygenated, non-acidic, and aqueous. Under these conditions, extracellular iron is predominantly found in the poorly soluble ferric (oxydized Fe3+) state. One way that organisms such as yeast improve iron bioavailability is by acidifying the local environment.  By lowering the pH of the surrounding environment, organisms facilitate solubilization and uptake of iron. ATP-driven proton transporters move H+ ions from the cytosol across the plasma membrane to control the pH at the cell surface.

Question: Had this system not have to emerge fully setup right from the beginning in order to facilitate and make Iron uptake into the cell even possible ?

Many microorganisms, including some fungi, also secrete low molecular weight compounds known as siderophores into their surroundings, which form high-affinity (~10−33 M) complexes with ferric iron to make it bioavailable for uptake. Transporters on the cell surface then recapture the Fe3+-siderophores complexes.  

Siderophores
Many organisms produce siderophores that bind iron extracellularly and that are subsequently transported together with the iron into the cell. Nitrogenase contains iron as a cofactor and also the electron donor to nitrogenase, ferredoxin, requires iron. 47 Siderophores are low-molecular weight, high-affinity Fe(III)-binding ligands secreted by bacteria under conditions of iron stress to scavenge and transport iron. In order to confine iron from solid minerals of marine aswell as freshwater environments (e.g., iron oxide hydrates), stones and rocks etc., siderophores must recognize, bind and sequester iron from solid minerals.   46 Siderophores bind to Fe3+ to form a ferrisiderophore complex which facilitates the transport of ferric ions into cells. In an aerobic, neutral-pH environment, the concentration of free Fe3+ is limited to 10-18 M by the insolubility of Fe(OH)3; this concentration is well below that generally required by cells. 45 Many microorganisms circumvent this nutritional limitation by producing siderophores (siderous= iron, phorus= bearer), low-molecular-weight compounds secreted under iron-limited conditions. These chelating agents strongly and specifically bind, solubilize, and deliver iron to microbial cells via specific cell surface receptors.

Siderophores are small molecular iron chelators that are produced by microbes and whose most notable function is to sequester iron from the host and provide this essential metal nutrient to microbes. 43 Currently, there are almost 500 compounds that have been identified as siderophores. Although siderophores differ widely in their overall structure, the chemical natures of the functional groups that coordinate the iron atom are not so diverse. Siderophores incorporate either α-hydroxycarboxylic acid, catechol, or hydroxamic acid moieties into their metal binding sites  and thus can be classified as either 

- hydroxycarboxylate 
- catecholate
- hydroxamate

type siderophores  44  The three broad groups are distinguished by the chemical structure of the metal-binding functionality (Figure below)

Iron Uptake and Homeostasis in Prokaryotic Microorganisms BEhgArC
(Top) Principal functional groups found in siderophores for Fe3+ coordination. 
The three functional groups are bidentate because each has two oxygen atoms that coordinate iron. (Bottom) The octahedral coordination of Fe3+ occurs by having six ligand atoms in a close-packed geometry around the metal ion. A schematic view of the pseudo-octahedral geometry of Fe3+ in siderophores composed of bidentate chelating units (such as hydroxamate and catecholate) is shown.

Iron Uptake and Homeostasis in Prokaryotic Microorganisms DVGfKjv

Siderophores bind to Fe3+ to form a ferrisiderophore complex which facilitates the transport of ferric ions into cells during periods of iron deficiency. Schizokinen, a citrate derivative siderophore was first reported from the freshwater cyanobacterium Anabaena PCC 7120 in 1983.

Iron Uptake and Homeostasis in Prokaryotic Microorganisms VrBB73B
Functional groups found in siderophores. 
Although the structures of siderophores may vary, the functional groups for Fe3+ coordination are limited. Siderophores usually contain the following metal-chelating functional groups: 
(a) α-hydroxycarboxylic acid, 
(b) catechol, or 
(c) hydroxamic acid. Each functional group is bidentate in that two oxygen atoms are involved in coordinating the iron atom. Since Fe3+ prefers a hexa-coordinate octahedral ligand sphere, three of these groups would make up an ideal Fe3+ binding site. Since Fe3+ is a hard metal ion it also prefers hard ligands, like oxygen. Fe2+ is a borderline metal ion that prefers tetrahedral coordination and softer ligands such as nitrogen. Therefore, reduction of Fe3+ to Fe2+ drastically lowers the affinity of the siderophore for the metal ion and causes its release. A siderophore structure from each chemical class are shown as follows: 
(d) hydroxamate siderophore ferrichrome, 
(e) the catecholate siderophore enterobactin, 
(f) the mixed catecholate-hydroxamate siderophore anguibactin, and 
(g) the hydroxycarboxylate siderophore rhizoferrin.

Although structurally diverse, most siderophores have some common features, including hard donor atoms (usually oxygen but occasionally nitrogen or sulfur) and the formation of thermodynamically stable, high-spin Fe3+ species.

The consequence of an oxidized atmosphere is that Iron is turned into scarcely soluble Fe3+ oxide hydrates d. To cope with this problem many organisms release iron chelating e compounds, called siderophores, which make iron available more or less specifically to the producing species. Cyanobacteria produce catecholate as well as hydroxamate siderophores, but nothing is known about their structures with the exception of the citrate-hydroxamate, schizokinen. Interestingly, schizokinen is also produced by other types of bacteria, which according to the secular evolutionary narrative suggests that the ability to produce this substance is either inherited from a common ancestor or has been acquired, by convergent evolution or gene transfer. 40

The mechanisms of siderophore biosynthesis involves a series of elongating acyl-S-enzyme intermediates on multimodular protein assembly lines, large multimodular enzymes: nonribosomal peptide synthetases (NRPS) 5  The biosynthesis of these products is carried out in a stepwise assembly from the amino acid monomers. Each module is dedicated to the activation, optional modification, and incorporation of one monomer into the product and harbors all necessary enzymatic activities in form of specialized domains to perform the single chemical reactions. 14 A substantial variety of siderophore structures are produced from similar NRPS assembly lines, and variation can come in the choice of the phenolic acid selected as the N-cap, the tailoring of amino acid residues during chain elongation, the mode of chain termination, and the nature of the capturing nucleophile of the siderophore acyl chain being released. Of course the specific parts that get assembled in a given bacterium may reflect a combination of the inventory of biosynthetic and tailoring gene clusters available. This modular assembly logic can account for all known siderophores. 39

Siderophores are the strongest Fe(III) chelators secreted by microorganisms and plants. 41 Siderophore production and secretion occurs, especially under iron starvation, when the intracellular iron concentration drops under a certain threshold required for functionality. Depending on the chemical nature of the organic ligand that coordinates iron, siderophores can be divided into following classes: the catecholates, and the hydroxamates or mixed-types that contain another iron complexing group such as α-hydroxy-carboxylate next to the hydroxamate catecholate group. Once bound to Fe(III), the ferrisiderophore is transported into the  cell via specific transporters on the cell surface. The hydroxamate-type siderophore schizokinen is a derivative of citric acid and chelates iron via two α-hydroxamate groups and one α-hydroxy-carboxylate group

Iron Uptake and Homeostasis in Prokaryotic Microorganisms WhZ18fr
Siderophore secretion by cyanobacteria is an intriguing process. The only cyanobacterial siderophore secretion pathway studied in detail, so far, is in Anabaena sp. PCC 7120. 

Nonribosomal peptide synthetases (NRPS) 
Nonribosomal peptide synthetase (NRPS) modular biosynthetic enzymes are responsible for the production of a multitude of structurally diverse and biologically important small molecule natural products. Traditional biochemical and genetic studies of these enzymes have contributed substantially to the understanding of their underlying biosynthetic mechanisms. Recalling that there was virtually no knowledge of these classes of enzymes 50 years ago, we must admire the vision of the scientists involved in the initial studies and mechanistic elucidation of carrier-protein-mediated biosynthesis, on whose shoulders the current field stands.  63

Iron Uptake and Homeostasis in Prokaryotic Microorganisms Wf7qhRv
Timeline of some notable events in the study of carrier-protein-mediated FAS, PKS, and NRPS biosynthetic enzymes

In the early 1990s, several independent laboratories discovered genes encoding the world’s biggest enzymes: the PKSs, which build polyketide chains k from simple acyl-CoA building blocks, and the the nonribosomal peptide synthetases (NRPs)  , which construct peptides from amino acids. The current world record holder, a PKS named MlsA1, weighs in at 1.8 million Daltons l, contains over 16,000 amino acids. The products of catalysis by these gigantic enzymes had already been known for decades. Science motivated efforts to decipher how these systems work and to understand why and how nature uses big proteins. 

The biosynthesis of nonribosomal peptides (NRPs)  is carried out by nonribosomal peptide synthetases (NRPSs), large, multidomain enzymes commonly arranged in co-linear assemblies of repeating modules. The linked domains orchestrate multiple and diverse chemical reactions in a highly coordinated manner. 36

Nonribosomal peptide synthetases (NRPSs) are a family of microbial megaenzymes that produce natural products like siderophores amongst other products. They are modular enzymes that catalyze synthesis of important peptide products from a variety of standard and non-proteinogenic amino acid substrates. Within a single module are multiple catalytic domains that are responsible for incorporation of a single residue. After the amino acid is activated and covalently attached to an integrated carrier protein domain, the substrates and intermediates are delivered to neighboring catalytic domains for peptide bond formation or, in some modules, chemical modification. In the final module, the peptide is delivered to a terminal thioesterase domain that catalyzes release of the peptide product. 23 They deliver amino acid and peptide intermediates, covalently bound to the pantetheine cofactor of a peptidyl carrier protein ( PCP ), to different catalytic domains where the nascent peptide chain is elongated, modified, and ultimately released. The primary catalytic domains are the adenylation domain. They  are modular enzymes that contain multiple catalytic domains joined as a single, multidomain protein.  Many bacteria use large modular enzymes for the synthesis of  peptide natural products like siderophores. These multidomain enzymes contain integrated carrier domains that deliver bound substrates to multiple catalytic domains, requiring coordination of these chemical steps. 25 They are macromolecular machines with modular assembly-line logic, a complex catalytic cycle, moving parts and multiple active sites. 19 They produce myriad life-saving natural products including potent antibiotics (e.g., penicillin) and anticancer drugs. These hundreds-of-kilodalton enzymes are composed of distinct domains in a molecular assembly line manner, and each domain is responsible for catalyzing a distinct chemical transformation with a controlled timing to construct natural peptidyl products of remarkable chemical complexity 35 The reaction sequence of nonribosomal peptide synthesis on a NRPS template is highly specific and leads to a single product. The reaction sequence catalyzed by peptide synthetases is highly template dependent, yielding a product of a distinct length and order. Omitting one amino acid substrate results in a total breakdown of product formation, indicating that initiation of peptide synthesis cannot occur at internal modules of the magasynthetase complexes. 37 

Ribbon diagrams of complete NRPS modules below: 

Iron Uptake and Homeostasis in Prokaryotic Microorganisms 9F6766h

(a) Domain architecture of three structurally characterized termination modules. b–d, The protein structures of 
(b) AB3403, 
(c) EntF, and 
(d) SrfA-C are coloured with domains coloured white (condensation), pink and red (adenylation domain N- and C-terminal subdomains), green-cyan (PCP), and blue (thioesterase). The phosphopantetheine moieties of AB3403 and
EntF, and inhibitor Ser-AVS, are highlighted.


Nonribosomal peptides and polyketides are biosynthesized with remarkable fidelity, typically as a single major product after tens to hundreds of enzymatic reactions. The fidelity of nonribosomal peptide synthetases (NRPSs)  comes from what is called the “thiotemplate mechanism” or “assembly-line enzymology.” 38

The modular  nonribosomal peptide synthetases (NRPSs) are among the largest and most complicated enzymes in nature. 22   In these biosynthetic systems, independently folding protein domains, which are organized into units called ‘modules’, operate in assembly-line fashion to construct polymeric chains and tailor their functionalities. Beginning in the 1990s, structural biology has provided a number of key insights. The emerging picture is one of remarkable dynamics and conformational programming in which the chemical states of individual catalytic domains are communicated to the others, configuring the modules for the next stage in the biosynthesis. This unexpected level of complexity most likely accounts for the low success rate of empirical genetic engineering experiments and suggests ways forward for productive megaenzyme synthetic biology.

 NRPSs are modular multienzymes. The immense size of the  NRPS polypeptides derives from their underlying division-of-labor organization (Figure below).

Iron Uptake and Homeostasis in Prokaryotic Microorganisms SyzTDML
Biosynthetic cycles within PKS and NRPS modules. 
(a) Schematic of minimal NRPS initiation and chain extension modules, in which the domain acting at each stage is indicated by a broken border. Biosynthesis begins with activation of a specific amino acid building block as its aminoacyl adenylate by an A domain (i) and transfer to a peptidyl carrier protein (PCP) (ii). The C domain then catalyzes peptide bond formation using the two aminoacyl-PCPs as substrates. This sequence of reactions is repeated until off-loading of the full-length polypeptide by the terminal TE domain. Chemical modification by tailoring domains such as N-methyltransferases and epimerases (not shown) can occur either before or following chain extension. 
(b) Schematic of minimal PKS initiation and chain extension modules. The AT domains initiate chain building by selection of an appropriate acyl-CoA (i) and transfer to the ACP of the same module (ii). The ketosynthase of the first extension module self-acylates with the starter unit (iii), which it then condenses with the extender unit attached to the ACP within its own module. Following chain extension, the β-keto group can be reductively modified by KR, DH and ER domains (not shown). As in NRPSs, release of the chain from the multienzyme is catalyzed by a TE.

Iron Uptake and Homeostasis in Prokaryotic Microorganisms ZYTWoZN

Type I NRPS responsible for tyrocidine production. 
( Tyrocidine is a mixture of cyclic decapeptides produced by the bacteria Bacillus brevis found in soil. It can be composed of 4 different amino acid sequences, giving tyrocidine A–D ) 64

In fact, these biosynthetic enzymes are assembly lines on a molecular scale: the growing chains are handed off from one specialized catalytic domain to the next, with each site performing a specific function. The domains are joined together via so-called ‘linker’ regions and grouped into functional units called modules. The first module initiates the biosynthesis, whereas the others extend the growing chain by one building block and chemically tailor its functionality. When the construction process is over, the product is liberated from the assembly line and then undergoes further processing reactions to attain its final bioactive form. A single NRPS polypeptide or ‘subunit’ typically contains multiple modules (MlsA1 has nine), and, with few exceptions, each pathway incorporates several subunits. 

In general, an NRPS catalytic cycle  starts with the selection and ATP-dependent activation of amino acids by adenylation (A) domains, which are then transferred onto the phosphopantetheine (P-pant) prosthetic group of peptidyl carrier protein (PCP) domains. The residues may then be modified by N-methyltransferase (N-MT) domains before being joined together to form peptides by catalytic condensation (C) domains . Further structural adjustments may occur after chain extension, such as epimerization (by E domains), formation of five-membered heterocyclic rings (heterocyclization (HC) domains) and oxidation (Ox domains). The appropriately processed peptide is then passed along to the next modules in line for further cycles of extension and tailoring before being released from the assembly line by a thioesterase (TE) domain, often in cyclic form. 

Although for all these systems it is possible to identify the chemical steps they carry out to arrive at the observed product structures, such models shed little light on how the multienzymes actually operate. The complication with  NRPS systems is not only that multiple catalytic sites, which may be located on the same or different polypeptides, work in series but also that the substrates are tethered to carrier proteins. These features result in a set of mechanistic issues unique to assembly line systems: how the catalytic domains communicate with the carrier proteins and how these interactions are regulated, the balance between enzyme-substrate and protein-protein interactions in programming the biosynthesis, how the multiple subunits recognize their correct partners and avoid incorrect associations are yet questions to be answered. 

NRPSs are macromolecular machines with modular assembly-line logic, a complex catalytic cycle, moving parts and multiple active sites. 19 They are organized into repeating sets of domains, called modules. Each module contains all functionality to introduce a building block into the growing peptide.  

Iron Uptake and Homeostasis in Prokaryotic Microorganisms Pu5cxD6
Nonribosomal peptides and peptide synthetases. 
(a) Some examples of nonribosomal peptides: gramicidin A (topical antibiotic), cyclosporin A (immunosuppressant), enterobactin (siderophore), yersinibactin (siderophore), bacillamide D (anti-algael), daptomycin (antibiotic, trade name Cubicin). 
(b) A schematic diagram of the elongation cycle of a canonical NRPS elongation module. 
(c) A generic synthetase. In this review, we follow the synthesis of a hypothetical formylated dipeptide that would be synthesized by the F–A–PCP–(C–A–PCP)n–Te NRPS depicted (where n = 1; note that most NRPSs do not contain an F domain, the tailoring domain which formylates the N-terminal amino acid). The domains included in some important crystal structures discussed in this review are indicated below the schematic. (d) Some examples of NRPSs. (ArCP: aryl carrier protein domain; Cy: heterocyclization domain; E/C: bifunctional epimerization/condensation domain.).

Dissecting the modules into catalytic domains
NRPSs consist of an arrangement of modules. A module is defined as a section of the NRPS’s polypeptide chain that is responsible for the incorporation of one building block into the growing polypeptide chain.

Iron Uptake and Homeostasis in Prokaryotic Microorganisms 4u28hAe
Coming from the gene, modules that are responsible for the incorporation of one amino acid can be identified on the protein level. Modules can be subdivided into domains that harbor the catalytic activities for substrate activation (A-domain), covalent loading (CP-domain), and peptide bond formation (C-domain). Modules lacking a C domain are used to initiate nonribosomal peptide synthesis, while those harboring a C-domain qualify for elongation.

Thus, NRPSs are used simultaneously as template (because the amino acid to be incorporated is determined by the module) and biosynthetic machinery (it is the module that harbors all necessary catalytic functions). Because fungi often use a single NRPS to synthesize a natural product, these multifunctional enzymes can reach remarkable sizes. For instance, Tolypocladium niveum uses a single, 1.6 MDa NRPS containing 11 modules to synthesize cyclosporine A. In bacteria, modules are usually distributed over several NRPSs whose genes are organized in an operon. Syringomycin synthetase E (Pseudomonas syringae) is currently the largest bacterial NRPS known, comprising a total of 8 modules. 33 The enzymatic units that reside within a module are called domains (Figure above). These domains catalyze at least the steps of substrate activation, covalent binding, and peptide bond formation of nonribosomal peptide synthesis . Domains of equal function share a number of highly conserved sequence motifs. These “core-motifs” allow the identification of individual domains on the protein level.

The Functional Domains of Modular Peptide Synthetases
NRPSs use a modular architecture with multiple catalytic domains joined as a single protein. Most commonly, each module adds one amino acid to the nascent peptide. Within a module are peptidyl carrier protein (PCP) domains that are posttranslationally modified with the phosphopantetheine group of coenzyme A. 59 

Synthesis of Coenzyme A
http://reasonandscience.catsboard.com/t2691-synthesis-of-coenzyme-a

Bound to the pantetheine through a thioester linkage, amino acid and peptide intermediates are delivered to adjacent catalytic domains in an assembly line fashion. Upstream of the PCP domains are adenylation domains that load the amino acid onto the pantetheine cofactor. The loaded amino acids serve as substrates for condensation domains that catalyze peptide bond formation. This process continues until the peptide is released by a thioesterase (TE) domain of the termination module. Although this linear architecture is sometimes used for the complete peptide, many NRPS clusters use several multidomain proteins, requiring both intra- and intermolecular domain interactions for the complete synthesis.

The adenylation (A) domain, the peptidyl carrier protein (PCP) domain and the condensation (C) domain are the three core domains that define a minimal module (with the exception of the first module, which usually lacks a C domain) 49

NRPSs typically synthesize their products through amide bond formation between aminoacyl (or other acyl) monomers. Their architecture is dissimilar to the more famous peptide maker. Whereas ribosomes use the same active sites for each amino acid added to the ribosomal peptide, NRPSs typically employ a dedicated set of enzyme domains for each amino acid added to the nonribosomal peptide. This set of domains is termed a module, and the synthetic strategy dictates that, normally, the number and specificity of the modules correspond to the length and sequence of amino acids in the peptide product. NRPSs can consist of a single polypeptide of between 2 and 18 modules, or be split over multiple proteins that assemble non-covalently (Figure above) Within a module, the domains work together to incorporate the incoming amino acid into the growing peptide. A basic elongation module contains 

- a condensation (C) domain, 
- an adenylation (A) domain, and 
- a peptidyl carrier protein (PCP) domain. 

The A domain selects and adenylates the cognate amino acid, then attaches it by a thioester link to a prosthetic phosphopantetheinyl (PPE) group on the PCP domain. The PCP domain then transports the amino acid to the C domain, which catalyzes amide bond formation between this amino acid and the peptide attached to the PCP domain of the preceding module, elongating the peptide by a single residue. Next, the PCP domain brings the elongated peptide to the downstream module, where it is passed off and further elongated in the next condensation reaction. Once a PCP domain has donated its peptide, it can accept a new amino acid from the A domain and participate in the next cycle of assembly-line synthesis. Initiation modules lack the C domain, and termination modules usually contain a thioesterase (Te) domain, which releases the peptide by cyclization or hydrolysis. A canonical organization of a basic NRPS is A–PCP-(C– A–PCP)n–Te (Figure c above). Additionally, NRPS modules very often have tailoring domains, including oxidase, reductase, epimerization, ketoreductase, aminotransferase and methyltransferase domains, and the action of these domains is incorporated into the catalytic cycle of the module. NPRSs can alternatively end in a reductase or terminal C domain. This wide range of tailoring domains, combined with the over five hundred monomers that can be used as substrates, including D-amino acids, aryl acids, hydroxy acids, and fatty acids, allows nonribosomal peptides to occupy a diverse area of chemical space.

The assembly of a nonribosomal peptide by an NRPS involves a series of repeating steps that are catalyzed by the coordinated actions of catalytic domains.  The structural features of putative domains and potentially important residues that might be involved in substrate specific 
 
- A: Adenylation  (A domain) (required in a module. Activates a cognate amino acid)
- PCP: Thiolation (T domain) and Peptide Carrier Protein with attached 4'-phospho-pantetheine (required in a module. Transfers the covalently attached aminoacyl motif along the assembly line)
- C: Condensation (C domain) catalyzes and formes the amide bond (required in a module)
- TE: (TE domain) Termination by a thio-esterase (only found once in a NRPS)release of the thioester bound peptide chain  that cleaves the product from the protein template 24

Together, these  core domains comprise a  NRPS module. For type A, linear NPRSs, the number of modules indicates the number of amino acids incorporated into a nonribosomal peptide. A fourth domain, a thioesterase, is often found at the C-terminus of the NRPS and catalyzes the release of the peptide from the NRPS. 32

Complementing the core domains common to all NRPSs, additionally auxiliary domains catalyze various modifications of the peptide while embedded into the assembly line. The addition of further chemical modifications contributes significantly to the structural diversity of NRPs, adding functional diversity and in vivo stability. These modifications occur during peptide synthesis and are performed by domains such as

- E: Epimerization into D-amino acids (optional)
- Cy: Cyclization into thiazoline or oxazolines (optional)
- Ox: Oxidation of thiazolines or oxazolines to thiazoles or oxazoles (optional)
- NMT: N-methylation (M domain) (optional)
- F: Formylation (optional)
- Red: Reduction of thiazolines or oxazolines to thiazolidines or oxazolidines (optional)
- R: Reduction to terminal aldehyde or alcohol (optional)

responsible for the incorporation of D-amino acids, heterocyclic rings, and N-methylated residues respectively. Incorporation of nonproteinogenic D-amino acids in the peptide framework is, in particular, a common modification for bioactive nonribosomal peptides 36 

PCP: Thiolation ( T- Domain )
All of the biosynthetic strategies involving Nonribosomal peptide synthetases (NRPSs)  are mediated by the Peptidyl carrier proteins (PCPs) domain, which is also referred to as the T domain - thiolation domains, found in each NRPS module.  Peptidyl carrier proteins (PCPs)  are central, and play a critical role  in nonribosomal peptide synthetase (NRPS)  enzymology, where the CPs are referred to as  peptidyl-carrier proteins (PCPs) or  Acyl-Carrier Proteins (ACPs) respectively ( structure, see below ) 38  

Iron Uptake and Homeostasis in Prokaryotic Microorganisms Y2hX8LP

Solution structure of peptidyl carrier protein PCP,The four helices are shown along with Ser44, the site of phosphopantetheinylation.

The peptidyl-carrier proteins (PCPs) is an important component of NRPSs biosynthesis with the growing chain bound during synthesis as a thiol ester f at the distal thiol of a 4'-phosphopantetheine moiety. The protein is expressed in the inactive apo form and the 4'-phosphopantetheine moiety must be post-translationally attached to a conserved serine residue on the ACP by the action of holo-Acyl-Carrier Proteins (ACPs), a 4'-phosphopantetheinyl transferase. 5

Due to its spatial neighborhood to the peptidyl carrier protein (also called thiolation domain T) in NRPSs, the aminoacyl residue of the adenylate is transferred to the sulfhydryl residue of the prosthetic group 40-phosphopantetheine (Ppant). Thereby the energy rich bond is conserved in the thioester formed. The prosthetic group is attached to the protein at an evolutionarily invariant serine 67

Iron Uptake and Homeostasis in Prokaryotic Microorganisms ZSlUq20

Crystal structure of the 4'-phosphopantetheinyl transferase sfp-coenzyme a complex 
In molecular biology, the 4'-phosphopantetheinyl transferase superfamily of proteins transfer a 4'-phosphopantetheine (4'-PP) moiety from coenzyme A (CoA) to an invariant serine in an acyl carrier protein (ACP), a small protein responsible for acyl group activation 65  4′-Phosphopantetheinyl transferases (PPTase) post-translationally modify carrier proteins with a phosphopantetheine moiety, an essential reaction in all three domains of life.

Iron Uptake and Homeostasis in Prokaryotic Microorganisms BJ5Ht2h
Phosphopantetheine, also known as 4'-Phosphopantetheine, is an essential prosthetic group of acyl carrier protein (ACP) and peptidyl carrier proteins (PCP) and aryl carrier proteins (ArCP) derived from Coenzyme A 66

Iron Uptake and Homeostasis in Prokaryotic Microorganisms SGdxgzH

Carrier protein posttranslational modification by coenzyme A. 
Carrier protein CP is pictured as a helical bundle, with cartoon representation in parentheses. A PPtase mediates transfer of the 4' -phosphopantetheine prosthetic group to a conserved serine residue of the carrier protein (pictured in red), converting it from apo to holoform. The terminal thiol can then be used to tether intermediates throughout  NRPS biosynthesis


So, we have following players here: 

Peptidyl carrier proteins (PCPs) ( or also called acyl carrier protein (ACP) )
4'-phosphopantetheine moiety (ppant)
4'-phosphopantetheinyl transferase (PPTase)

NRPS peptidyl-carrier proteins (PCPs) are ~80–95 amino acid long proteins that are posttranslationally modified with a 4′-phosphopantetheinyl (ppant) group from coenzyme A by phosphopantetheinyl transferases (PPTase), also known as holo-ACP or holo-PCP synthases. All carrier proteins have a conserved serine which requires 4′-phosphopantetheinylation by a PPTase in order to tether cargo via a flexible, labile, thioester linkage.

The Phosphopantetheinyl Transferases: Catalysis of a Posttranslational Modification Crucial for Life 53
2015 Jan 1
Phosphopantetheinyl transferases (PPTases) are essential for cell viability across all three domains of life: bacteria, archaea and eukaryota. PPTases posttranslationally modify modular and iterative synthases acting in a processive fashion, namely fatty acid synthases (FAS), polyketide synthases (PKS), and non-ribosomal peptide syntethases (NRPS). The central component of these chain elongating synthases is non-catalytic and  independently translated protein. Regardless, this protein component is referred to as a carrier protein (CP), or alternatively a thiolation domain. CP is responsible for timing and efficiency in shuttling the rapidly changing chemical intermediates due to chain elongation between the structurally diverse multienzyme complexes of these pathways. The CP tethers the growing intermediates on a 4′-phosphopantetheine (PPant) arm of  through a reactive thioester linkage. PPants are thought of as “prosthetic arms” ( attached arm ) on which all substrates and intermediates of these pathways are covalently yet transiently held during the orderly progression of enzymatic modifications to the extending chain. PPTases mediate the transfer and covalent attachment of PPant arms from coenzyme A (CoA) to conserved serine residues of the CP domain through phosphoester bonds. These essential posttranslation protein modifications convert inactive apo-synthases to active holo-synthases (Figure below).

Iron Uptake and Homeostasis in Prokaryotic Microorganisms UvGy1xj
General reaction scheme of post-translational phophopantetheinylation by a PPTase
The PPTase transfers the PPant moiety ( part of the molecule) from CoA to a conserved serine residue on the apo-CP to produce holo-CP, here showcased by a typical NRPS module containing C, condensation; A, adenylation; and CP, carrier protein, domains. 3′, 5′-PAP is 3′,5′-phosphoadenosine phosphate. h


PPTases are essential for many bioactive secondary metabolites, and a variety of other central biosynthetic pathways in both primary and specialized metabolism. Many of these PPTases have been described on the gene and protein levels providing for intense biochemical characterization. With the mapping of their active sites, their interactions and catalytic mechanisms accompanying CoA and CP recognitions provided quantitative clarity, in some cases delineating predictable strategies for their molecular engineering for an assortment of basic and applied applications.

NRPS are either assembled from one or more multi-domain polypeptides. Carrier Proteins of NRPSs are referred to as peptidyl carrier proteins (PCPs). Conserved serine residues of the CPs must be functionalized by PPTase catalysis with a PPant arm.

Post-translational modification of peptide synthases 55
1997
Phosphopantetheinyl transferases (PPTases) catalyze the post-translational modification of proteins by the covalent attachment of the 4’-phosphopantetheine (P-pant) moiety of coenzyme A (CoASH) to a conserved serine residue of the protein substrate, a reaction with mechanistic analogies to the widely studied serine phosphorylation post-translational modification catalyzed by the hundreds of protein serine kinases. 


Iron Uptake and Homeostasis in Prokaryotic Microorganisms WGJ6ZmI

Reaction catalyzed by PPTases. 
PPTases transfer the moiety of coenzyme A onto the side chain hydroxyl group of a serine residue in ACPs and PCPs to convert them from their inactive apo- into the active holo-form. 56

Without proper post-translational addition of P-pant, the carrier protein is nonfunctional, effectively killing the activity of the polyketide synthase or peptide synthetase.

Substrate specificity is conferred on multiple levels, including the enzymatic active sites and specific interactions between the CPs and catalytic domains.  In a typical round of peptide bond formation a PCP must interact with an A-domain and the acceptor site of an upstream condensation domain (C-domain) and acceptor donor site of a downstream C-domain.

The PCP domain is relatively small (approximately 80–100 amino acid), and functions as a central tether for the amino acid building blocks and the growing peptidyl intermediates. 34  To function as a covalent tether, a PCP domain
must be post-translationally modified with a prosthetic 4-phosphopantetheine (Ppant) group. The Ppant arm is attached to a conserved Ser residue of the PCP domain by associated phosphopantetheinyl transferases (PPTases) in a Mg2+-dependent reaction involving Coenzyme A as a substrate which converts the inactive apo-PCP to active holo-PCP. 

Iron Uptake and Homeostasis in Prokaryotic Microorganisms IGpyw4T
PCP modification by coenzyme A and PPTases. 
A PPTase catalyzes the attachment of the Ppant prosthetic group to a conserved serine residue of the PCP; converting it from its inactive apo-form to its active holo-form.

The NRPS enzymes use a peptidyl carrier protein (PCP) domain that is used to shuttle the substrates and peptide intermediates between different catalytic domains. The PCP domains are the smallest NRPS domains, usually only 70–90 amino acids in length. 57   The PCP domains contain a conserved serine residue that serves as the site for covalent modification with a phosphopantetheine cofactor that is derived from coenzyme A (Figure below)

Iron Uptake and Homeostasis in Prokaryotic Microorganisms YVEvdyM
Chemical structure of the phosphopantetheine cofactor attached to a conserved serine residue of the peptidyl carrier protein.




a The thioesterases (TEs), or thioester hydrolases, comprise a large enzyme group whose members hydrolyze the thioester bond between a carbonyl group and a sulfur atom.  16 Substrates of 15 of these 27 groupings contain coenzyme A (CoA), two contain acyl carrier proteins (ACPs), four have glutathione.

b The simplest amides are derivatives of ammonia wherein one hydrogen atom has been replaced by an acyl group. 20 

Iron Uptake and Homeostasis in Prokaryotic Microorganisms EZdis6E

Iron Uptake and Homeostasis in Prokaryotic Microorganisms V4mrUpw

An acyl group is a moiety derived by the removal of one or more hydroxyl groups from an oxoacid 21

Iron Uptake and Homeostasis in Prokaryotic Microorganisms YgMVz7I

c Adenylation, also known as adenylylation or AMPylation, is the process of attaching an AMP molecule to a protein side chain by covalent bonding. It has two main functions: 1) to regulate enzyme activity via post-translational modification and 2) to produce unstable intermediates of a protein, peptide or amino acids to allow reactions that are not thermodynamically favored to occur. 29  Adenosine monophosphate (AMP), also known as 5'-adenylic acid, is a nucleotide. AMP consists of a phosphate group, the sugar ribose, and the nucleobase adenine; it is an ester of phosphoric acid and the nucleoside adenosine. 30

d Iron oxides are chemical compounds composed of iron and oxygen. All together, there are sixteen known iron oxides and oxyhydroxides 42

e the Amino Acid must be activated. This involves the addition of ATP (adenosine triphosphate), forming Aminoacyl Adenylate.

 Iron Uptake and Homeostasis in Prokaryotic Microorganisms RMRhlef

f In chemistry thioesters are compounds,  products of esterification between a carboxylic acid and a thiol. Esterification is the general name for a chemical reaction in which two reactants (typically an alcohol and an acid) form an ester as the reaction product. Esters are common in organic chemistry and biological materials, and often have a characteristic pleasant, fruity odor. This leads to their extensive use in the fragrance and flavor industry. Ester bonds are also found in many polymers. 50

Iron Uptake and Homeostasis in Prokaryotic Microorganisms VKoFHRH

g The donation of electrons from an electron-rich portion of a molecule (this can be a lone pair, or even electrons already involved in bonds) to an electron-poor part of either the same, or a different molecule. The electron rich “donor” is called a nucleophile, and the electron poor “acceptor” is called an electrophile. This “attack” or “donation” usually results in the formation of a bond between the nucleophile and electrophile.

Iron Uptake and Homeostasis in Prokaryotic Microorganisms PtPCidP
Arrows are usually drawn to indicate the direction of “attack”. In this case the electrons are coming from the :Nu and going to the C+. Thus the :Nu is “attacking” the C+, and acting as a nucleophile.

h  3'-Phosphoadenosine-5'-phosphosulfate (PAPS) is a derivative of adenosine monophosphate that is phosphorylated at the 3' position and has a sulfate group attached to the 5' phosphate. It is the most common coenzyme in sulfotransferase reactions. It is endogenously synthesized by organisms via the phosphorylation of adenosine 5'-phosphosulfate (APS), an intermediary metabolite. 54 

i Thiols are the sulfur analogue of alcohols (that is, sulfur takes the place of oxygen in the hydroxyl group  j of an alcohol), and the word is a portmanteau of "thion" + "alcohol," with the first word deriving from Greek theion = "sulfur". 60

j A hydroxy or hydroxyl group is the entity with the formula OH. It contains oxygen bonded to hydrogen. In organic chemistry, alcohol and carboxylic acids contain hydroxy groups. The anion [OH−], called hydroxide, consists of a hydroxy group. 61  A carboxylic acid is an organic compound that contains a carboxyl group. The general formula of a carboxylic acid is R–COOH, with R referring to the rest of the (possibly quite large) molecule. Carboxylic acids occur widely and include the amino acids (which make up proteins) and acetic acid (which is part of vinegar and occurs in metabolism). 62

k Polyketides are a large group of natural products produced by microorganisms and plants. They are biopolymers of acetate and other short carboxylates and are biosynthesized by multifunctional enzymes called polyketide synthases (PKSs). 68

l The unified atomic mass unit or dalton (symbol: u, or Da) is a standard unit of mass that quantifies mass on an atomic or molecular scale (atomic mass). One unified atomic mass unit is approximately the mass of one nucleon (either a single proton or neutron) and is numerically equivalent to 1 g/mol 69

1. Binding, Transport and Storage of Metal Ions in Biological Cells, page 329
2. https://www.ncbi.nlm.nih.gov/pubmed/8592731
3. http://journal.frontiersin.org/article/10.3389/fphar.2014.00173/full
4. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3924584/
5. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC120789/
6. https://en.wikipedia.org/wiki/Evolution_of_metal_ions_in_biological_systems
7. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4303624/
8. Calcium homestasis, page 9
9. Practical Approaches to Biological Inorganic Chemistry, page 16
10. https://en.wikipedia.org/wiki/Nonribosomal_peptide
11. http://sci-hub.hk/https://www.sciencedirect.com/science/article/pii/S0959440X10000126
12. http://sci-hub.hk/https://www.sciencedirect.com/science/article/pii/S1074552196901817
13. https://static-content.springer.com/esm/art%3A10.1038%2Fs41598-017-12244-3/MediaObjects/41598_2017_12244_MOESM1_ESM.pdf
14. http://www.pnas.org/content/99/22/14083.full
15. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2854587/
16. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2974821/
17. http://sci-hub.hk/http://pubs.rsc.org/en/content/articlelanding/2016/np/c4np00148f/unauth#!divAbstract
18. http://sci-hub.hk/http://pubs.rsc.org/en/content/articlelanding/2010/np/b912037h/unauth#!divAbstract
19. http://sci-hub.hk/https://www.sciencedirect.com/science/article/pii/S0959440X17301161
20. https://en.wikipedia.org/wiki/Amide
21. https://en.wikipedia.org/wiki/Acyl_group
22. http://sci-hub.hk/https://www.nature.com/articles/nchembio.1883
23. Nonribosomal Peptide and Polyketide Biosynthesis  Methods and Protocols, page 3
24. http://sci-hub.hk/https://pubs.acs.org/doi/pdf/10.1021/cr960029e
25. http://sci-hub.hk/https://pubs.acs.org/doi/abs/10.1021/bi300112e
26. https://www.ncbi.nlm.nih.gov/books/NBK22356/
27. http://sci-hub.hk/http://pubs.rsc.org/en/Content/ArticleLanding/2007/NP/b603921a#!divAbstract
28. http://sci-hub.hk/http://pubs.rsc.org/en/content/articlelanding/2012/np/c2np20025b#!divAbstract
29. https://en.wikibooks.org/wiki/Structural_Biochemistry/Proteins/Adenylation
30. https://en.wikipedia.org/wiki/Adenosine_monophosphate
31. http://sci-hub.hk/https://pubs.acs.org/doi/abs/10.1021/cb900156h
32. http://sci-hub.hk/https://pubs.acs.org/doi/abs/10.1021/mp700137g
33. http://sci-hub.hk/https://www.annualreviews.org/doi/pdf/10.1146/annurev.micro.58.030603.123615
34. http://sci-hub.hk/http://www.ingentaconnect.com/content/ben/coc/2015/00000019/00000013/art00003?crawler=true
35. http://sci-hub.hk/https://pubs.acs.org/doi/abs/10.1021/bi501156m
36. http://sci-hub.hk/https://pubs.acs.org/doi/abs/10.1021/acschembio.6b00332
37. http://sci-hub.hk/https://pubs.acs.org/doi/abs/10.1021/bi000768w
38. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4109001/
39. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC120789/
40. https://www.degruyter.com/downloadpdf/j/znc.2000.55.issue-9-10/znc-2000-9-1002/znc-2000-9-1002.pdf
41. Genomics of Cyanobacteria, page 91
42. https://en.wikipedia.org/wiki/Iron_oxide
43. http://mbio.asm.org/content/7/6/e01906-16.full
44. http://sci-hub.hk/https://www.sciencedirect.com/science/article/pii/S0005273607002738
45. http://sci-hub.hk/http://www.asmscience.org/content/book/10.1128/9781555816544.chap1
46. http://sci-hub.hk/https://www.sciencedirect.com/science/article/pii/S073497500900055X
47. Stressbiology of cyanobacteria, page 301
48. http://sci-hub.hk/http://www.biochemj.org/content/445/3/297
49. http://www.microbiologyresearch.org/docserver/fulltext/micro/150/6/1629.pdf?expires=1519934963&id=id&accname=guest&checksum=9A990433F60527D2F315FC297497979C
50. https://en.wikipedia.org/wiki/Ester#Preparation
51. http://emboj.embopress.org/content/16/14/4174
52. http://sci-hub.hk/https://www.nature.com/articles/nature16163
53. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3918677/
54. https://en.wikipedia.org/wiki/3%27-Phosphoadenosine-5%27-phosphosulfate
55. http://sci-hub.tw/10.1016/S1367-5931(97)80067-1
56. http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6968.2002.tb11285.x/pdf
57. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4760355/
58. https://en.wikipedia.org/wiki/Acyl_carrier_protein
59. http://sci-hub.tw/https://www.sciencedirect.com/science/article/pii/S1074552112000063
60. https://en.wikipedia.org/wiki/Thiol
61. https://en.wikipedia.org/wiki/Hydroxy_group
62. https://en.wikipedia.org/wiki/Carboxylic_acid
63. http://sci-hub.tw/http://pubs.rsc.org/en/content/articlelanding/2009/cs/b805115c/unauth#!divAbstract
64. https://en.wikipedia.org/wiki/Tyrocidine
65. https://en.wikipedia.org/wiki/4%27-phosphopantetheinyl_transferase
66. https://en.wikipedia.org/wiki/Phosphopantetheine
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69. https://en.wikipedia.org/wiki/Unified_atomic_mass_unit



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This post-translational modification converts the apo-carrier protein to a holo- state and is catalyzed by a specific phosphopantetheinyl transferase (PPTase). The thiol of the phosphopantetheine group binds covalently to the amino acid and peptide substrates through a thioester linkage with the carboxyl group of the amino acid.   Helices 1, 2, and 4 are longer, and mostly parallel, while the third helix is shorter and runs approximately perpendicular to the axes of the other three. The serine residue that is the site of addition of the phosphopantetheine group is located at the start of helix α2. This helix is preceded by a long loop that is diverse in sequence and structure between the different NRPS PCP domains. 

The remarkable feature of carrier proteins from any system is the specificity with which they interact with other proteins. 27 Maintaining the proper three dimensional shape of the carrier protein domain, or retaining the ability to distort conformation in the case of PCP, is essential, as the 4-phosphopantetheine arm must remain available for interaction with all the relevant modification domains. However, carrier proteins at the N or C terminus of multimodular PKS and NRPS systems must interact with other proteins in order to complete their products. Recent work has elucidated domains that are involved in conferring specificity in the interactions between multi-domain polypeptides. These short (20 amino acid) domains are found attached to carrier proteins or any domain that is located at the interacting termini of a synthase protein. The first protein that carrier proteins must interact with is the 4-phosphopantetheinyl transferase (PPTase).

A new enzyme superfamily - the phosphopantetheinyl transferases 12 
22 October 1996
All non-ribosomal peptide synthetases require posttranslational modification of their constituent acyl carrier protein domain(s) to become catalytically active. The inactive apoproteins are converted to their active holo-forms by posttranslational transfer of the 4’-phosphopantetheinyl (P-pant) moiety of coenzyme A to the sidechain hydroxyl of a conserved serine residue in each acyl carrier protein domain. Multienzyme complexes exist for acyl group activation and transfer reactions in the biogenesis of almost all non-ribosomal peptides. All of these complexes contain one or more. small proteins, -80-100 amino acids (aa) long, either as separate subunits or as integrated domains, that function as carrier proteins for the growing acyl chain. This acyl carrier protein (ACP) domains, which may be one of the domains of a multi-functional enzyme (in the type I synthases) or a separate subunit (in the type II multienzyme complex synthases), can be recognized by the conserved sequence signature motif.



Iron Uptake and Homeostasis in Prokaryotic Microorganisms A4zPu8k

Schematic representation of post-translational phosphopantetheinylation of a CP domain by a PPTase. 13

The priming reaction, in which the ppant-arm is attached covalently to a highly conserved serine residue (GGXS-motif), is carried out by ppant-transferases.  Approximately 80% of coenzyme A (CoA), the precursor of the ppant cofactor, is acetylated in bacteria, thus mispriming events during the Sfp-mediated PCP phospantetheinylation occur quite regularly, which interrupt the NRP assembly. These dead end misprimed PCP species are repaired by the action of so-called type II thioesterases (TEII). 

The Adenylation step
NRPS Adenylation   domains play a key role in peptide natural product biosynthesis. They select, activate, and transfer amino acids to carrier protein domains. 2 The activation of the amino acid is analogous to the reaction that loads tRNAs for ribosomal protein synthesis. An aminoacyl adenylate  a   is formed by attack upon the a-phosphate of ATP b , and the activated amino acid is then transferred to the free thiol terminus of the phosphopantetheinyl arm of a peptidyl carrier protein. Because of its role in identifying the correct amino acid to append to the carrier protein, the A domain  is the gatekeeper for ensuring that the correct units are incorporated into the growing peptide chain.  A domains must be able to differentiate between a vast number of possible substrates. In A domains, residues that line the active site confer the amino acid specificity. By analyzing sequences of all the known A domains and using the pocket of the phenylalanine specific A domain as a model, eight variable residues were identified. These residues can be used to predict the specificity of A domains that have not been characterized. 

In the assembly line-like choreography, the adenylation domain is the first domain the substrate encounters before it is added to the nascent peptide natural product. 1  The A domain (~550 amino acid) found in all NRPS modules is an essential catalytic component that also functions as a gatekeeper for the selection of amino acid building blocks during NRP biosynthesis. 34 The A domain selectively incorporates cognate amino acids into peptide-based natural products from a much larger monomer pool, including all 20 proteinogenic amino acids as well as a number of non-proteinogenic amino acids, aryl acids, fatty acids, and hydroxy acid building blocks. The A domain initially recognizes a cognate amino acid and converts it to the corresponding aminoacyl adenylate intermediate at the expense of Mg2+ and a molecule of ATP with the release of pyrophosphate (PPi) ( figure b below). The adenylated substrate subsequently undergoes nucleophilic attack g  by the terminal thiol group of the Ppant arm of a downstream PCP domain, leading to the formation of a thioester bound aminoacyl-S-PCP ( see figure b below )

Iron Uptake and Homeostasis in Prokaryotic Microorganisms UCHr8gy
(b) Adenylation reaction catalyzed by the A domain


The A domain recognizes the amino acid substrate and activates it first through the formation of an aminoacyl adenylate e and then via covalent binding of the activated amino acid as a thioester f to the phosphopantetheinyl (49Ppant) cofactor of the PCP domain This cofactor is posttranslationally attached to a conserved serine of PCP domains ( see chapter PCP domain, above  )by a phosphopantetheinyl transferase. Elongation of the peptidyl chain is then carried out by the last essential domain, the C domain ( See figure a below )

Substrate recognition by A domains
A domains activate and transfer amino acids to PCP domains (Figure a below) and are thus considered to be the primary determinant of substrate selectivity. 

Cocrystallization of PheA with L-Phe and AMP allowed the identification of the hydrophobic L-Phe-binding pocket and of the residues making contact with Phe (Fig. b below)

Iron Uptake and Homeostasis in Prokaryotic Microorganisms Wn4mOD2
A domains of NRPS. 
(a) Reactions catalysed by A domains: the amino acid substrate is activated through the formation of an aminoacyl adenylate followed by the covalent binding via a thioester bond to the Ppant cofactor of the PCP domain. 
(b) Structure of the L-phenylalanine ( an α-amino acid )  L-Phe-activating domain (PheA) of the gramicidin S synthetase GrsA, with an enlargement of the L-Phe-binding pocket showing the residues of PheA making contact with L-Phe. The side chains of these residues and the substrate L-Phe are rendered in ball and stick representation. 49

The study of their specificity has been greatly facilitated by the determination  of the crystal structure of the A domain of the gramicidin S synthetase GrsA, the L-Phe-activating domain PheA, solved in 1997 in following paper: 

Structural basis for the activation of phenylalanine in the non-ribosomal biosynthesis of gramicidin S 51
15.07.1997
The non‐ribosomal synthesis of the cyclic peptide antibiotic gramicidin S is accomplished by two large multifunctional enzymes, the peptide synthetases 1 and 2. The enzyme complex contains five conserved subunits of ∼60 kDa which carry out ATP‐dependent activation of specific amino acids and share extensive regions of sequence similarity with adenylating enzymes such as firefly luciferases and acyl‐CoA ligases. We have determined the crystal structure of the N‐terminal adenylation subunit in a complex with AMP and L‐phenylalanine to 1.9 Å resolution. The 556 amino acid residue fragment is folded into two domains with the active site situated at their interface. Each domain of the enzyme has a similar topology to the corresponding domain of unliganded firefly luciferase, but a remarkable relative domain rotation of 94° occurs. This conformation places the absolutely conserved Lys517 in a position to form electrostatic interactions with both ligands. The Adenosine monophosphate (AMP) is bound with the phosphate moiety interacting with Lys517 and the hydroxyl groups of the ribose forming hydrogen bonds with Asp413. The phenylalanine substrate binds in a hydrophobic pocket with the carboxylate group interacting with Lys517 and the α‐amino group with Asp235. The structure reveals the role of the invariant residues within the superfamily of adenylate‐forming enzymes and indicates a conserved mechanism of nucleotide binding and substrate activation. 

Each of the five modules in which the grs operon is organized encodes for highly conserved functional subunits. The major one is a 60 kDa fragment which recognizes a specific amino acid and catalyses the adenylation of the amino acid carboxylate group with the α‐phosphate of ATP. This adenylation subunit is conserved not only within all known peptide synthetases, but also shares extensive sequence similarity with firefly luciferases and acyl CoA ligases. Common to all these enzymes is the ATP‐dependent activation of substrates as acyl adenylates.

The adenylation subunit shares no sequence homology with enzymes involved in the ribosomal synthesis of polypeptides, despite the fact that the formation of aminoacyl‐adenylates is chemically analogous in the two systems.

This is a remarkabe example of convergence on a molecular level, where the same biological function is achieved by different routes.

Description of the overall structure
The polypeptide chain folds into two compact domains (Figure below). There are very few direct protein–protein interdomain contacts and instead the interactions between the structural domains are mediated by a network of hydrogen bonds between the side chains of the protein and a sandwiched layer of ordered water molecules. The much larger N‐terminal domain comprising residues 17–428 contains three subdomains: a distorted β‐barrel and two β‐sheets which pack together to form a five‐layered αβαβα tertiary structure (Figure 2). Subdomain A contains a six‐stranded β‐sheet and three helices formed by a single segment of the polypeptide chain (residues 91–203) while a seventh strand is formed by an insertion in the β‐barrel subdomain (Figure 3). The β‐sheet B contains eight strands, of which the first two (B1–B2) are formed by residues occurring before β‐sheet A in the polypeptide chain, while the remaining six strands (B3–B8) and four helices form a contiguous polypeptide segment located before the β‐barrel subdomain in the sequence. Strands 1–6 in the two β‐sheets share a similar topology, with strands A1–A4 in sheet A corresponding to strands B3–B6 in sheet B while strands A5–A6 correspond to strands B1–B2.


Iron Uptake and Homeostasis in Prokaryotic Microorganisms Bt7cin8
Ribbon diagram of the PheA molecule 
The large N‐terminal domain shown in blue and the small C‐terminal domain in green. The disordered loop (residues 192–196) near the active site is coloured violet. The AMP (red) and phenylalanine (orange) ligands are drawn using a space‐filling representation. The side chain of Lys517 on the loop that projects down from the C‐terminal domain is drawn in green using a ball‐and‐stick representation.

Several siderophore biosynthetic gene clusters have been identified and their biochemical behaviors have been studied in vitro, and the results of these studies have expanded our knowledge of the biosynthesis of several aryl-capped peptide siderophores.

In 1997, the first X-ray crystal structure of the A domain of the peptide synthetase GrsA from the gramicidin S biosynthetic pathway was determined in a complex with L-Phe and AMP ( see figure below ) 

Iron Uptake and Homeostasis in Prokaryotic Microorganisms N8v9E8o
(a) Crystal structure of the L-Phe-activating A domain of gramicidin S synthetases from Aneurinibacillus migulanus. The L-Phe and AMP have been shown as stick structure, which are colored magenta and black, respectively. The large N-terminal domain and the smaller C-terminal domain are colored cyan and green, respectively.
(b) Crystal structure of the aryl acid adenylating enzyme DhbE of the siderophore bacillibactin synthetases from Bacillus subtilis [43]. The large N-terminal domain and the smaller C-terminal domain are colored cyan and green, respectively. 
(c) Schematic summary of the substrate binding pocket from the phenylalanineactivating A domain of GrsA.

Following on from this work, the discrete DHB-activating A domain (DhbE) of the bacillibactin biosynthetic pathway was solved in 2002 (Figure b above). The A domains of NRPS enzymes generally display 30–60% sequence identity and share a highly conserved three-dimensional structure. These adenylating enzymes share several common structural features, including a large N-terminal subdomain and a small C-terminal subdomain, with the active site itself being placed at the junction between these two domains. All NRPS A domains exhibit high sequence identity, making the A domain structures of GrsA and DhbE prototypes for all of the amino acid- and aryl acid-activating A domains in NRPSs, respectively. Sequence alignments of the residues lining the active sites of various NRPS A domains and the results of structurefunction mutagenesis studies have allowed for a series of general rules pertaining to the structural factors underpinning substrate recognition, activation, and selectivity processes in NRPS A domains to be strictly defined. As the results, some 8–10 amino acid residues have been identified as the nonribosomal peptide codes (Table below).

Iron Uptake and Homeostasis in Prokaryotic Microorganisms MYYVHog
The specificity-conferring codes of the A domains found in NRPSs.

Figure c above describes a simplified representation of the proposed binding pocket of the phenylalanine-activating A domain of GrsAThese rules can be used to engineer the substrate specificity of NRPS A domains and predict their substrate specificity as well as the chemical structures of secondary metabolites resulting from the identified NRPS genes.

The initial step of chain extension is the selection and activation of a specific amino acid by A domains using ATP to convert the amino acid to an aminoacyl-AMP intermediate, followed by transfer of the activated amino acid on to the thiol moiety of the phosphopantetheine (Ppant),posttranslationally-modified PCP domain forming an aminoacyl-S-Ppant-PCP. 36  The activation of various nonproteinogenic amino acid building blocks (>500 precursors7) by adenylation domains is achieved via a reactive acyl-adenylate - the adenylation step ( See figure below ) 35

Iron Uptake and Homeostasis in Prokaryotic Microorganisms UIQBRws

on/off Pathways of the Reactive Phenylalanyl Adenylate Intermediate for L-Phe Activation. This intermediate then forms a thioester bond with the free thiol group at the 4′-phosphopantetheine (Ppant) in the PCP domain, which is the the thioesterification step, facilitating  the downstream substrate channeling10 at C, TE, and other possible tailoring domains.

The A-domain (∼550 aa) is responsible for the selection of the amino acids that make up the product and thus controls its primary sequence. A-domains activate amino or carboxy acid substrate as amino acyl adenylate while ATP is consumed. Two crystal structures of A-domains have been solved to date. The crystal structure of the phenylalanine-activating A-domain of the gramicidin S-synthetase A, GrsA from Bacillus brevis, allowed the assignment of those amino acid residues that play a decisive role in the coordination of the substrate.

The three-domain initiation module PheATE (GrsA) of Bacillus breVis gramicidin S synthetase catalyzes the 

- activation, 
- thiolation and 
- epimerization 

of L-phenylalanine (L-Phe), the first amino acid incorporated into the decapeptide antibiotic gramicidin S. There are three activated intermediates in the PheATE catalyzed chemical pathway: 

- L-phenylalanyl-adenosine-5′-monophosphate diester (L-Phe-AMP), 
- L-Phe-S-4′-phosphopantetheine(Ppant) 
- D-Phe-S-4′-Ppant-acyl enzyme.

The cofactor-independent epimerization reaction of gramicidin S synthetase (GrsA_PheATE) is a deprotonation/reprotonation mechanism, most likely through an peptidyl enolate intermediate.

These residues lie in a 100-aa stretch between cores A4 and A5 of the A-domain. A more exact analysis of this region within different A-domains led to the introduction of the so-called nonribosomal code, which allows the prediction of A-domain selectivity on the basis of its primary sequence. This code was initially restricted to amino acid–activating A-domains but was extended to carboxy acid–activating A-domains when the crystal structure of the 2,3-DHB-activating A-domain DhbE from B. subtilis was solved. In addition to the prediction of A-domain selectivity, the nonribosomal code was also recently exploited for the creation of two A-domains with altered selectivity. 

Despite the similarity in enzymatic activity of adenylate-forming enzymes, Adomains and aa-tRNA synthetases are structurally unrelated.

So we have here a nice example of convergent functions, achieved by different routes. 

The substrate binding pocket is located at the interface between the two subunits. In contrast to a model in which the subunits undergo a structural rearrangement during catalysis, two recently solved crystal structures  reveal a different mechanism . The DhbE structures show three snapshots of catalysis, that is, 

- without substrate, 
- with bound DHB and AMP, and 
- with the product DHB-adenylate. 

Only minor structural movements that are restricted to the so-called p-loop (phosphate binding loop, or Walker A motif) take place during catalysis. The p-loop is located at the entrance of the catalytic pocket, which it covers during catalysis presumably to shield ATP from the surrounding water and to initiate catalysis.

Following is the description of the structure of both the holo and apo forms of the PCP-E didomain of the gramicidin S synthetase GrsA, the first module of the cyclic peptide antibiotic (gramicidin S) synthetase. The biosynthesis of gramicidin S in Bacillus brevis is catalyzed by the NRPS GrsA and GrsB; in which, five modules incorporate ten amino acids cyclized into the final cyclic peptide. 

Iron Uptake and Homeostasis in Prokaryotic Microorganisms HFF0dgl
Gramicidin S synthetase assembly line. 
a) Cartoon representation of the NRPS enzymes, GrsA and GrsB. The amino acid building blocks are selected and activated by A domains (adenylation domain, blue, activated amino acid shown in subscript). PCP domains
(peptidyl carrier protein, orange) use the 4’-phosphopantethiene, post-translational modification to transport acyl-intermediates. C domains (condensation, green) catalyze peptide bond formation between acyl-S-PCP intermediates of adjacent modules. The E domain (epimerization domain, grey) produces D-phenylalanine (the epimerized stereocenter is highlighted, red) through epimerization at the α-carbon. The di-domain fragment described in this study is shown in an orange box. Gramicidin S 
b) is released from the termination module by a cyclization reaction of the dimeric pentapeptide catalyzed by the Te domain (thioesterase domain, brown).

Iron Uptake and Homeostasis in Prokaryotic Microorganisms G2KgSe3

Overview of the GrsA_PCP-E di-domain fragment. 
a) The overall V-shape structure of  the E subdomains characteristic of the chloramphenicol-acetyltransferase (CAT) fold is shown in  grey, the PCP domain is shown in yellow, the linker connecting PCP and E domain is colored in orange, and the bridge region is colored blue. The electron density (Fo-Fc map is displayed at  1.6σ contour level) around the Ppant arm is shown as green mesh (green). 
b) Detailed view of  the Ppant arm (in a ball and stick configuration) located at the center of the V-shaped E domain.  The proposed catalytic residues, H753 and E892 are shown in red. A highly conserve tyrosine  residue (976) interacts with the gem-dimethyl group of the Ppant arm. 
c) Schematic of the major  interactions of GrsA_PCP-E residues with the Ppant arm, the hydrogen bonds are shown as  blue-dashed lines with H894, K945, and Y976, and the hydrophobic pocket is created by I574,  I866, and Y976.

Iron Uptake and Homeostasis in Prokaryotic Microorganisms CP6Qz2m

Overview of GrsA_PCP-E surface and domain/domain interactions.  The E domain is shown in grey, the PCP domain is shown in yellow, and the linker is in orange. 
a) The interface between the linker and E domain (green). (A1) Detailed view of contact area created by E and linker regions. The linker is shown in cartoon representation with positively charged arginines colored with blue as stick. Negative charged residues on E domain are colored with red. 
b) The interface between PCP and E domain. The contact areas on the E domain is colored blue, and the n PCP domain, red. (B1) Detailed view of interface created by E and PCP domain. The PCP domain is shown in cartoon presentation with key interacting residues colored red.

NRPS adenylation  domains play a key role in peptide natural product biosynthesis.  In the assembly line-like choreography, the adenylation domain is the first domain the substrate encounters before it is added to the nascent peptide natural product. The adenylation domains catalyze a two-step reaction that activates the amino acyl substrate as an adenylate, followed by transfer of the amino acid to the thiol of the pantetheine cofactor of the carrier protein domain:

Iron Uptake and Homeostasis in Prokaryotic Microorganisms KjjAEzM
Reaction catalyzed by the NRPS adenylation domain ( A domain ) 

Adenylation domains belong to a larger adenylate-forming enzyme superfamily containing Acyl-CoA synthetases, NRPS adenylation domains, and beetle luciferase. The ANL superfamily of adenylating enzymes contains acyl- and aryl-CoA synthetases, firefly luciferase, and the adenylation domains of the modular non-ribosomal peptide synthetases (NRPSs). 31 Members of this family catalyze two partial reactions: the initial adenylation of a carboxylate to form an acyl-AMP intermediate, followed by a second partial reaction, most commonly the formation of a thioester. Recent biochemical and structural evidence has been presented that supports the use by this enzyme family of a remarkable catalytic strategy for the two catalytic steps. The enzymes use a 140° domain rotation to present opposing faces of the dynamic C-terminal domain to the active site for the different partial reactions. The adenylation domains of non-ribosomal peptide synthetases (NRPSs)  catalyzes the activation of a carboxylate substrate with ATP to form an acyl adenylate intermediate that is used in a diverse set of second partial reactions.

Adenylation (A) Domains. 
The A domains catalyze a two-step, ATP-dependent reaction that involves the activation of the carboxylate group of the amino acid (or for some initiating substrates, aryl acid) substrate as an aminoacylAMP intermediate and subsequent transfer of the amino acid to the 4′-Ppant of the neighboring thiolation domain (Figure A)

Iron Uptake and Homeostasis in Prokaryotic Microorganisms Gguh5jy
Schematic representations of the reactions catalyzed by each of the core domains of NRPSs.
The domain involved in the reaction shown at the right is highlighted in black on the left: 
(A) adenylation domain, catalyzing aminoacyl-AMP formation; 
(B) thiolation domain, highlighting the formation of the aminoacyl thioester; 
(C) condensation domain, showing the formation of a peptide bond between two aminoacylthioester substrates; the T1 and T2 denote the T domains from neighboring NRPS modules; the “d” and “a” shown within the black C domain denote the donor and acceptor sites, respectively; 
(D) thioester domain, catalyzing first aminoacylester formation on the Te domain followed by either hydrolysis or cyclization of the peptide. The “X” represents either a nitrogen or oxygen. The 4′-Ppant cofactor is represented by the SH bonded to each T domain. From this figure forward, 4′-Ppant cofactor will be represented in this manner.

A domains are generally referred to as the “gate keepers” of NRPSs because they select the amino acid to be activated and incorporated into the nonribosomal peptide and are therefore the first level of substrate selectivity in the enzyme system.  One reason for the enormous structural diversity of nonribosomal peptides is that A domains are not limited to the standard 20 proteinogenic amino acids. In fact, more than 500 different precursors have been identified in nonribosomal peptides. 

Crystal structures of two A domains have been determined to date; one activates L-Phe and is a representative of amino acid-activating A domains, while the other activates 2,3- dihydroxybenzoic acid and represents aryl acid-activating A domains. By defining an A domain substrate specificity code, it is now possible to predict what particular amino acid a given NRPS module incorporates based on the structure of the nonribosomal peptide.

The first step is the formation of an aminoacyl adenylate from an amino acid and ATP. This activated species is a mixed anhydride in which the carboxyl group of the amino acid is linked to the phosphoryl group of AMP; hence, it is also known as aminoacyl-AMP. 26 The A domain recognizes and activates the amino acid building block by formation of an amino acyl adenylate intermediate through the consumption of ATP. 28 The adenylation domains (A domain) represent the central points of action in multifunctional peptide synthetases. For each incorporated amino acid in the peptide product a specific adenylation domain exists, whose location also dictates the primary structure of the peptide product. Hence, investigations on peptide synthetases have notably focused on the A domain in recent years. 


Activation Reaction 
In order to incorporate an amino acid residue into a peptide through the protein template a two-step mechanism for substrate activation is required.

Iron Uptake and Homeostasis in Prokaryotic Microorganisms KUwfkMF
A simplified scheme displaying the principles of the thiotemplate-directed nonribosomal peptide synthesis. 
(A) The synthesis of the cyclic decapeptide gramicidin S on the multifunctional enzymes GrsA (one amino acid-activating module plus epimerization domain) and GrsB (four amino acid-activating modules) is shown. Each module (symbolized by a circle) activates the cognate amino acid by ATP hydrolysis as amino acyl adenylate. This relatively instable intermediate is stabilized by thioesterification on the cofactor 4′-PP. Thioesterified substrates are then integrated into the growing peptide through a step-by-step condensation. The amino acid-activating modules are arranged in the order that corresponds to the amino acid sequence of the peptide. The arrows indicate the direction of polymerization. 
(B) Structure of the peptide antibiotic gramicidin S. The cyclic decapeptide was obtained by a head-to-tail condensation of two identical pentapeptides synthesized by the protein template described above.

First, the cognate amino acid is activated as aminoacyl-adenylate at the expense of Mg2+-ATP (Figure below).

Iron Uptake and Homeostasis in Prokaryotic Microorganisms PhlY92f
Amino acid adenylation in peptide synthesis 

Second, the enzyme-attached thiol moiety 4′-phosphopantetheine (4′-PP) attacks the aminoacyl adenylate to yield the aminoacyl thioester and AMP as leaving group. The second step of the reaction requires the presence of the thiolation domain (T domain)The way in which the amino acid residues are activated resembles that catalyzed by aminoacyltRNA synthetases in the ribosomal system of peptide synthesis. There, the cognate amino acid is also activated as aminoacyl adenylate and then becomes esterified onto the 2′- or 3′-OH of the 3′-nucleotide of the corresponding tRNA, which acts as the carrier of the activated amino acid. Despite these similarities shared by the ribosomal and nonribosomal system during amino acid activation, the enzymes involved have no similarity in primary and 3D structures.  Strikingly, there exist two classes of aminoacyl-tRNA synthetases, catalyzing the formation of aminoacyl adenylate, with fundamentally different folding topologies. The recently solved crystal structure of an adenylation domain of a peptide synthetase reveals that there is a third fold for the same reaction employed. 

Adenylation Domains of Peptide Synthetases Are Members of a Superfamily of Adenylate-Forming Enzymes
The conserved region of the A domain was identified by comparing several genes encoding peptide synthetases. The highly conserved A domains were found as repetitive blocks, the number of which coincides with the number of amino acids activated by the corresponding synthetase. These blocks, connected by regions now designated the condensation domains, represent what we call the minimal module, containing the A and the T domain. The A domain is about 550 amino acids in length. It shares significant homology with the family of acyl-CoA synthases and luciferases, which are about the same size. Since all these enzymes catalyze an analogous reaction, the adenylation of their carboxy substrates, they constitute a superfamily of adenylate forming enzymes A T domain connected to an A domain is exclusively found in peptide synthetases and is involved in the second part of the amino acid activation, the thiolation reaction

Nonribosomal peptides (NRP) are a class of peptide secondary metabolites, usually produced by microorganisms like bacteria and fungi. Nonribosomal peptides are also found in higher organisms, such as nudibranchs, but are thought to be made by bacteria inside these organisms. While there exist a wide range of peptides that are not synthesized by ribosomes, the term nonribosomal peptide typically refers to a very specific set of these.

Nonribosomal peptides are synthesized by nonribosomal peptide synthetases, which, unlike the ribosomes, are independent of messenger RNA. Each nonribosomal peptide synthetase can synthesize only one type of peptide. Nonribosomal peptides often have cyclic and/or branched structures, can contain non-proteinogenic amino acids including D-amino acids, carry modifications like N-methyl and N-formyl groups, or are glycosylated, acylated, halogenated, or hydroxylated. Cyclization of amino acids against the peptide "backbone" is often performed, resulting in oxazolines and thiazolines; these can be further oxidized or reduced. On occasion, dehydration is performed on serines, resulting in dehydroalanine. This is just a sampling of the various manipulations and variations that nonribosomal peptides can perform. Nonribosomal peptides are often dimers or trimers of identical sequences chained together or cyclized, or even branched.

Nonribosomal peptides are a very diverse family of natural products with an extremely broad range of biological activities and pharmacological properties. They are often toxins, siderophores, or pigments. Nonribosomal peptide antibiotics, cytostatics, and immunosuppressants are in commercial use. 10

Nonribosomal peptides are synthesized by one or more specialized nonribosomal peptide-synthetase (NRPS) enzymes. The enzymes are organized in modules that are responsible for the introduction of one additional amino acid. Each module consists of several domains with defined functions, separated by short spacer regions of about 15 amino acids. 11 The biosynthesis of nonribosomal peptides shares characteristics with the polyketide and fatty acid biosynthesis. Due to these structural and mechanistic similarities, some nonribosomal peptide synthetases contain polyketide synthase modules for the insertion of acetate or propionate-derived subunits into the peptide chain.

Nonribosomal peptide synthetases (NRPSs) are large multimodular biocatalysts that utilize complex regiospecific and stereospecific reactions to assemble structurally and functionally diverse peptides. 11  During this ribosome-independent peptide synthesis, catalytic domains of NRPS select, activate or modify the covalently tethered reaction intermediates to control the iterative chain elongation process and product release. Nonribosomal peptides NRPs are produced in the secondary metabolism of bacteria and fungi by the consecutive condensation of amino acids, which is achieved by large multimodular enzymes, nonribosomal peptide synthetases (NRPSs). Notably, this process is not limited to the 20 proteinogenic amino acids. Some 500 different monomers, including nonproteinogenic amino acids, fatty acids, and a-hydroxy acids, have been identified as building blocks for NRPs. The nonproteinogenic building blocks contribute to structural versatility of NRPs and are likely to contribute substantially to the observed biological activity.  NRPSs are composed of an array of distinct modular sections each of which is responsible for the incorporation of one defined monomer into the final peptide product.

Iron Uptake and Homeostasis in Prokaryotic Microorganisms KEW7wFG
(a) Examples of nonribosomally assembled, clinically approved peptides. Next to the compound the names (first line), the trade names (second line), and the biological activity are given (third line). 
(b) Simplified mechanism of nonribosomal peptide (NRP) synthesis. (1) The amino acid is activated as aminoacyl-AMP by the adenylation domain. (2) Transfer of the amino acid onto the PCP domain. (3) Condensation of PCP-bound amino acids. (4) Possibility of amino acid modifications, for example by epimerization domains. (5) Transesterification of the peptide chain from the terminal PCP onto the TE domain. (6) TE catalyzed product release by either hydrolysis or macrocyclization. The number of modification domains and modules is very variable. (c) Examples of single domain structures [20 ,26,45,46]. PDB accession codes are given in parenthesis. 

The identity and order of a module in an assembly line specifies: first, the sequence of monomer units activated and incorporated; second, the chemistry that occurs at each way station in the assembly line; and third, the
length and functionality of the product released from the distal end of the assembly line (Figure b above)

The modules can be further divided into catalytic domains. Three domains are ubiquitous in NRP synthesis and essential for peptide elongation. The domains are responsible for the

- activation of the amino acid (adenylation (A) domains),
- the propagation of the growing peptide chain (thiolation or peptidyl carrier protein (PCP) domains), and
- the condensation of the amino acids (condensation (C) domains).
- A fourth essential NRPS catalytic unit associated with product release is the thioesterase (TE) domain. The TE domain is located in the termination module and catalyzes peptide release by either hydrolysis or macrocyclization.

Each domain structure has been determined by either crystal or NMR structure elucidation (Figure c above);

Reaction cycles of peptidyl carrier and adenylation domains
A domain reaction cycles and are depicted in Figure below.

Iron Uptake and Homeostasis in Prokaryotic Microorganisms T5ZUe4u

PCP and adenylation domain cycle. 
The apo-PCP domain (left half, green) is converted to the holo-form by a ppant-transferase (e.g. Sfp, left half, cyan) by ppant attachment to the active site serine. Mispriming, for example with acetyl-ppant is repaired by the thioesterase type II (left half, orange). The correctly primed holo-PCP domain interacts with the A domain cycle where it gets loaded with an amino acid. After translocation of the amino acid by condensation, the holo-PCP can be loaded again. In the A domain cycle (right half), the small C-terminal subdomain (brown) of the A domain (red) traverses several states. In an open conformation, the A domain is able to bind the amino acid and ATP. The adenylation intermediate then generates the aminoacyl-AMP and pyrophosphate is cleaved off. The aminoacyl-AMP intermediate is protected from bulk solvent in a closed formation, which likely facilitates transfer onto the PCP domain (thiolation). 

In the first step of the PCP cycle, the carrier (80 amino acids), which is responsible for the transportation, propagation, and presentation of the aminoacyl or peptidyl substrates of the growing NRP chain, is primed post-translationally with its 4'-phospopantetheine (p-pant) cofactor. Central to this biosynthetic strategy and usually a component of each module are the peptidyl carrier protein (PCP) domains. PCPs bind the monomers and intermediates of the growing peptide chain as thioesters through the thiol moiety of their prosthetic group 4′-phosphopantetheine (4′p-pant) and facilitate on this 20-Å long tether their directed transport through the NRPS assembly line. Conversion of each PCP from the inactive apo form into the active holo form is performed by 4′PP transferases in a posttranslational reaction, referred to as the priming step, which uses CoA as the source of 4′PP. Only when all PCPs are equipped with a 4′PP from the priming step, the NRPSs can synthesize their dedicated products in cycles of initiation, elongation, and termination steps

Regeneration of misprimed nonribosomal peptide synthetases by type II thioesterases 14
June 27, 2002
Nonribosomal peptide synthetases (NRPSs) assemble structurally complex peptides from simple building blocks such as amino and carboxyl acids. Product release by macrocyclization or hydrolysis is catalyzed by a thioesterase domain that is an integrated part of the NRPS enzyme.

Polyketide synthase and non-ribosomal peptide synthetase thioesterase selectivity: logic gate or a victim of fate?
12th November 2014
Type 1, a/b hydrolase-like thioesterase (TE) domains are essential offloading enzymes, releasing covalently bound products from  non-ribosomal peptide biosynthetic complexes. 17 Common to the biosynthesis  is the covalent attachment of the growing compound through a thioester bond to a carrier protein (CP). Expedient offloading of these acyl- and peptidyl-CP intermediates is required to prevent stalling and to enable turnover of the biosynthetic pathways. Cleavage of completed acyl or peptidyl chain from the terminal CP is catalyzed by a twostep TE-mediated mechanism (Figure below)

Iron Uptake and Homeostasis in Prokaryotic Microorganisms XWWGFlb
TEs catalyze substrate offloading from CPs of  non-ribosomal peptide synthetases.Acyl and peptidyl chains bound to the Ppant arm of CPs are loadedonto the active site serine of a TE and then released via nucleophilic attack.

The release step can occur by attack of an exogenous nucleophile effecting hydrolysis or transesterification or by an intramolecular O-, N-, or C nucleophile, effecting macrolactonization, macrolactamization or Claisen-like condensation. Thus in addition to ensuring turnover of the pathway, TEs provide access to increased chemical diversity. Because of the importance of TE-mediated offloading in polyketide and non-ribosomal peptide biosynthesis. In the context of all offloading mechanisms for  non-ribosomal peptides, Du and Lou4 provided a comprehensive review that included TE-mediated offloading and effectively highlighted its breadth and importance in offloading.

Assembly line of the Surfactin non-ribosomal peptide synthetase

NRPS release mechanisms
Liangcheng Du and Lili Lou,  6th October 2009
Polyketides (PKs) and nonribosomal peptides (NRPs) are two large groups of natural products with remarkable structural diversity and biological activities 18 NRPs are biosynthesized through the thiotemplate mechanism, where the NRP chain is assembled on enzyme templates and the biosynthetic intermediates are covalently attached to the templates as thioesters. Polyketides PKs are assembled from small building blocks such as acetate and other short carboxylic acids by sequential decarboxylative condensations, and this process is catalyzed by polyketide synthases (PKSs). In bacterial type I PKSs, a module is generally responsible for one cycle of polyketide elongation and the associated modifications. A PKS module consists of a b-ketoacyl synthase (KS), an acyltransferase (AT), and an acyl carrier protein (ACP). Most PKSs also contain accessory domains, such as b-ketoacyl reductase (KR)dehydratase (DH)enoylreductase (ER), and methyltransferase (MT). Throughout the biosynthesis, the growing polyketide intermediates remain covalently attached to the ACP domain via the 40 -phosphopantetheine cofactor. Once the growing polyketide chain reaches its full length, it is released from the PKS. Typically, a thioesterase (TE) domain that is usually located at the end of the assembly line is responsible for this release.

NRPs are a family of structurally complex natural products that are mainly found in microorganisms. These products are 2–48 amino acid residues in length, usually with highly diverse structures. More than 300 amino acids, including D-configured, N-methylated, and other unusual non-proteinogenic residues, are found in the products.

The peptides form linear, cyclic, or branched cyclic structures with such modifications as acylation, glycosylation, or heterocyclizaton. These peptides are synthesized by a group of multifunctional mega-enzymes, nonribosomal peptide synthetases (NRPSs), using proteins as templates to direct the incorporation of amino acids. The NRPSs are arranged in a modular structure, in which each module is a relatively independent functional block that fulfills a cycle of peptide elongation. A module is consisted of several domains, which perform distinct functions, such as adenylation (A-domain), thiolation (T-domain or PCP for peptidyl carrier protein), and condensation (C-domain). Through ATP-hydrolysis, the A-domain activates a specific amino acid substrate as an aminoacyl adenylate, which subsequently is covalently linked to the 40 -phosphopantetheinyl cofactor of the PCP domain to form a thioester, aminoacyl-S-PCP. The C-domain then catalyzes the amide bond formation between two cognate aminoacyl-S-PCPs to produce a dipeptide. During the entire elongation process, the growing peptide intermediates remain covalently tethered to the PCPs. Additional domains have also been identified that are responsible for the modification of the peptide chain, such as an epimerization (E) domain for the conversion of L-amino acids to D-amino acids. A TE domain is usually found at the end of the NRPSs for the cleavage of the full-length peptide product from the enzyme to terminate the biosynthesis.

NRPs  use carrier proteins – PCPs – as biosynthetic workstations and choose  40 -phosphopantetheine prosthetic group for precursor activation and intermediate channeling.  NRPSs, amino acids are activated as amino acyl-S-PCPs. In polyketide biosynthesis, the C–C bond formation is mediated by the KS domain that catalyzes the nucleophilic attack of the acyl-S-ACP from the upstream module with a carbon nucleophile generated from its cognate acyl-S-ACP, mostly malonyl- or methylmalonylS-ACP. 

An aminoacyl-tRNA synthetase (aaRS or ARS), also called tRNA-ligase, is an enzyme that attaches the appropriate amino acid onto its tRNA. It does so by catalyzing the esterification of a specific cognate amino acid or its precursor to one of all its compatible cognate tRNAs to form an aminoacyl-tRNA. In humans, the 20 different types of aa-tRNA are made by the 20 different aminoacyl-tRNA synthetases, one for each amino acid of the genetic code. This is sometimes called "charging" or "loading" the tRNA with the amino acid. Once the tRNA is charged, a ribosome can transfer the amino acid from the tRNA onto a growing peptide, according to the genetic code. Aminoacyl tRNA therefore plays an important role in RNA translation, the expression of genes to create proteins. 3

b ATP is the primary energy currency of the cell. It has an adenosine backbone with three phosphate groups attached.

Iron Uptake and Homeostasis in Prokaryotic Microorganisms I9CQJc5

c Adenylation, also known as adenylylation or AMPylation, is the process of attaching an AMP molecule to a protein side chain by covalent bonding. It has two main functions: 1) to regulate enzyme activity via post-translational modification and 2) to produce unstable intermediates of a protein, peptide or amino acids to allow reactions that are not thermodynamically favored to occur. 29  Adenosine monophosphate (AMP), also known as 5'-adenylic acid, is a nucleotide. AMP consists of a phosphate group, the sugar ribose, and the nucleobase adenine; it is an ester of phosphoric acid and the nucleoside adenosine. 30

1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4760355/
2. http://sci-hub.tw/http://pubs.rsc.org/en/Content/ArticleLanding/2007/NP/b603921a#!divAbstract
3. https://en.wikipedia.org/wiki/Aminoacyl_tRNA_synthetase
10. https://en.wikipedia.org/wiki/Nonribosomal_peptide
11. http://sci-hub.hk/https://www.sciencedirect.com/science/article/pii/S0959440X10000126
12. http://sci-hub.hk/https://www.sciencedirect.com/science/article/pii/S1074552196901817
13. https://static-content.springer.com/esm/art%3A10.1038%2Fs41598-017-12244-3/MediaObjects/41598_2017_12244_MOESM1_ESM.pdf
14. http://www.pnas.org/content/99/22/14083.full
17. http://sci-hub.hk/http://pubs.rsc.org/en/content/articlelanding/2016/np/c4np00148f/unauth#!divAbstract
26. https://www.ncbi.nlm.nih.gov/books/NBK22356/
27. http://sci-hub.hk/http://pubs.rsc.org/en/Content/ArticleLanding/2007/NP/b603921a#!divAbstract
28. http://sci-hub.hk/http://pubs.rsc.org/en/content/articlelanding/2012/np/c2np20025b#!divAbstract
29. https://en.wikibooks.org/wiki/Structural_Biochemistry/Proteins/Adenylation
30. https://en.wikipedia.org/wiki/Adenosine_monophosphate

31. http://sci-hub.hk/https://pubs.acs.org/doi/abs/10.1021/cb900156h
34. The Cell Biology of Cyanobacteria, page 60
35. http://www.pnas.org/content/108/6/2184.full
36. www.mdpi.com/2075-1729/5/1/841/pdf
49. https://en.wikipedia.org/wiki/Carbon_monoxide
51. http://www.sciencedirect.com.https.sci-hub.hk/science/article/pii/S0005273607002738



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TE-mediated product release mechanisms 
The TE-mediated product release mechanisms are regarded as the ‘‘canonical’’ mechanisms for most  NPR biosyntheses. The 3-D structures of  NPRS TEs have been solved.  The TE domain is usually located at the C-terminus of NRPS assembly lines.TE belongs to the a/b-hydrolase superfamily, which includes lipases, proteases and esterases. They all have a conserved catalytic triad, Ser-His-Asp, to release the product. When the linear NRP chain assembled by NRPS reaches its full length, it is transferred from the last ACP/PCP to the hydroxyl of the active site Ser of TE to form a (peptidyl)acyl ester (Figure below).

Iron Uptake and Homeostasis in Prokaryotic Microorganisms 66xKuK8

The general mechanism for TE-catalyzed NRP release
 TE’s three residues in the catalytic triad, Ser-His-Asp, are shown. Note that an acyl-S-ACP, not a peptidyl-S-PCP, is used as an example to illustrate the triad-catalyzed product release. The carbonyl oxygen of the acyl-S-ACP is stabilized by the oxyanion hole provided by the amide backbone of the TE protein. Depending on the nature of the TE domain, the acyl chain of acyl-O-TE could be hydrolyzed to produce a linear product (I) or macrocyclized to produce a macrolactone/macrolactam (II)

The His residue, which is stabilized by the Asp residue, acts as the catalytic base to accept the proton from the hydroxyl of the catalytic Ser residue. The nucleophilic hydroxyl oxygen of the Ser residue attacks the carbonyl carbon of the (peptidyl)acyl thioester bound on PCP/ACP to yield a (peptidyl)acyl-O-Ser oxoester on TE. The negative charge developed on the thioester during the nucleophilic attack is probably stabilized by the putative oxyanion hole provided by the amide backbone of the enzyme. Depending on the nature of the TE, the TE-bound (peptidyl)acyl-O-Ser intermediate is then attacked either by an external nucleophile, typically water, that leads to a linear hydrolyzed product (Fig. I) or by an internal nucleophile, typically a hydroxyl or an amino group on the intermediate, that leads to a macrocycle (macrolide or macrolactam) (Fig. II).

As macrocyclization is both chemically challenging and essential for the biological activity of many polyketide and non-ribosomal peptides, TE-mediated macrocyclization was reviewed by Kohli and Walsh in 2002 and non-ribosomal pepitide TE-mediated macrolactamization by Kopp and Marahie in 2007. 

Enzymology of acyl chain macrocyclization in natural product biosynthesis
Rahul M. Kohli and Christopher T. Walsh  22nd November 2002
Polyketides and nonribosomal peptides  are biosynthesized by the ordered condensation of monomer building blocks, acyl-CoAs or amino acids, leading to construction of linear acyl chains. Many of these natural products are constrained to their bioactive conformations by macrocyclization whereby, in one of the terminal steps of biosynthesis, parts of the molecule distant in the constructed linear acyl chain are covalently linked to one another. Typically, macrocyclization is catalyzed by a thioesterase domain at the C-terminal end of the biosynthetic assembly line. Biologically active natural products must present the proper functionality in the precise orientation required for interaction with a molecular target.  For many diverse natural products, including polyketides and nonribosomally synthesized peptides, covalent constraints are selectively achieved in densely functional molecules by enzymatic cyclization of linear acyl chains. Polyketide natural products are assembled from acyl monomer units activated as thioesters. The fundamental chain elongation step is C–C bond formation mediated by attack of an enzyme-generated carbon nucleophile upon an upstream biosynthetic intermediate. In type I polyketide synthases (PKS) the elongated acyl chain is translocated from upstream to downstream carrier protein domains that contain the tethering thiol group. In type II PKS, the elongated acyl chain stays tethered to the same carrier protein while the acyl monomers are on distinct subunits and the chain elongation is iterative. Nonribosomal peptides are assembled by parallel logic to the type I PKS. Peptidyl chains grow by consecutive addition of activated aminoacyl monomer units. The fundamental chain elongation step is peptide bond formation and the elongated chain is translocated each time from upstream to downstream carrier proteins during chain elongation.


In both PKS and nonribosomal peptide synthetases (NRPS), once the acyl chain reaches its full length on the most downstream carrier protein, it has to be released from its covalent thioester tether. Typically, the most C-terminal domain in these protein assembly lines is a thioesterase (TE) domain whose role is to catalyze the chain disconnection reaction. While hydrolysis is one common product of enzymatic chain termination, intramolecular macrocyclization reactions are catalyzed by several PKS and NRPS terminal domains. 

Assembly-line enzymatic machinery: initiation, elongation and termination modules
The enzymatic catalysts that perform the macrocyclization reactions are the most downstream domains in the multidomain, modular assembly lines of polyketide synthases and nonribosomal peptide synthetases. To understand the function of these cyclases in release of the full-length polyketide and polypeptide chains we note some conserved features of the enzymatic logic and organization common to both type I PKS and NRPS assembly lines. The protein modules of NRPS assembly lines are organized into chain initiation, chain elongation and chain termination modules, proceeding from N-terminal to C-terminal modules, respectively (Figure below).

Generic organizational scheme of NRPS and PKS assembly lines. 
The domains involved in the assembly lines are diagrammed as boxes with function denoted at right. A module is defined by the domains dedicated to the incorporation and modification of a single building block. Each module contains a thiolation domain with a phosphopantetheine tether (–SH). The dotted line indicates that the modules are often strung together into large multi-domain, multimodule synthetase subunits where the sum of several subunits constitutes a full assembly line. Within individual modules, domains may be present which function to modify the building block being incorporated into the growing peptide or polyketide.

A second thioesterase of type II (TEII) encoded by a distinct gene associated with the NRPS cluster was previously shown by means of gene disruption to be important for efficient product formation.  Here we report the biochemical characterization of two such TEII enzymes that are associated with the synthetases of the peptide antibiotics surfactin (TEIIsrf) and bacitracin (TEIIbac). Both enzymes were shown to efficiently regenerate misacylated thiol groups of 4′-phosphopantetheine (4′PP) cofactors attached to the peptidyl carrier proteins (PCPs) of NRPSs.  the physiological role of TEIIs in nonribosomal peptide synthesis is the regeneration of misacylated NRPS, which result from the apo to holo conversion of NRPS enzymes because of the promiscuity of dedicated 4′PP transferases that use not only free CoA, but also acyl-CoAs as 4′PP donors.

Another component associated with most of the analyzed NRPS biosynthetic gene clusters is a gene predicted to code for a type II thioesterase. These enzymes are important for effective synthesis, because deletion of the genes led to a drastic reduction in product yields, but did not completely abolish it.  4′PP transferases were shown to accept as cosubstrates not only free CoA, but also various acyl-CoA derivatives like acetyl-CoA. In these cases, transfer of the corresponding acyl-4′PP onto PCPs would result in inactive NRPSs, because substrates without an α-amino group cannot be elongated. Indeed, a significant fraction of the CoA pool in the cell is in the form of various acyl-CoAs. We therefore carried out biochemical experiments to test the validity of both the cleaning after aminomisacylation and the deblocking after mispriming models. Comparison of the catalytic properties of a TEII to hydrolyze the thioester bond of aminoacyl- and peptidyl-PCP substrates on the one hand and of an acetyl-PCP substrate on the other hand showed a strong preference of the TEII for the latter and thus supports the deblocking after mispriming model.

The Condensation Domain
Condensation domains, usually located at the N-terminus of a module, catalyze amide bond formation between two substrates. The condensation domains transfer the amino acid or peptide from an upstream carrier protein domain to the amino moiety of the substrate that has been previously loaded onto a downstream carrier protein domain (Figure below). 1

Iron Uptake and Homeostasis in Prokaryotic Microorganisms YHajHcF
Reaction catalyzed by the NRPS condensation domain.

These 450 residue domains belong to the chloramphenicol acetyltransferase (CAT) superfamily. Similar to CAT, condensation domains contain a conserved HHxxxDG motif. In CAT, the second His of this motif acts a general base that extracts a proton from chloramphenicol promoting nucleophilic attack and thus acyl transfer. This His is also essential for condensation domain activity; however, its exact role may depend on the substrates. Currently there are four crystal structures of condensation domains. They are: the standalone condensation domain VibH, the final condensation domain and its donor PCP dissected from the multi modular TycC, the condensation domain in the terminal module SrfA-C solved as a complete module, and finally the first condensation domain dissected from Calcium-Dependent Antibiotic synthetase (CDA-C1).

The Thioesterase Domain
Within the final module of an NRPS pathway, the activity of the final condensation domain catalyzes the transfer of the upstream peptide to the amino acid substrate that is loaded onto the terminal PCP domain. To release the peptide and free the NRPS enzyme for another round of synthesis, the activity of a thioesterase domain is required (Figure below).

Iron Uptake and Homeostasis in Prokaryotic Microorganisms Jv7ub9e
Reaction catalyzed by the NRPS thioesterase domain.

The thioesterase domains are approximately 30 kD in size and, as a class, can function as either hydrolases (as shown in Figure above) or as cyclases, where they can catalyze either lactam or lactone formation with an upstream heteroatom from the peptide chain. The thioesterase domains form an acyl-enzyme intermediate with an active site serine residue that subsequently is released from the enzyme through either hydrolysis with a water nucleophile or cyclization. For enzymes that catalyze lactone or lactam formation, the active site pocket therefore must bind the peptide substrate in an orientation that favors cyclization over hydrolysis by positioning the nucleophilic group to resolve the acyl-enzyme intermediate.

1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4760355/



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The amazing efforts of cells to import, transport, homeostatic control and  iron processing for the manufacture of protein cofactors 

http://reasonandscience.catsboard.com/t2443-iron-uptake-and-homeostasis-in-prokaryotic-microorganisms#5913

The following narrative will exemplify, how many ultracomplex steps that are required, many different and functionally varied molecular machines, nano-factory-like assembly lines, complex molecular  interactions, proofreading mechanisms, error excision and repair, and machine-like procedures, just to get one life-essential element,  Iron, to be recruited, imported, transported, its right intracellular homeostatic level controlled, and synthesized  to make cofactors and electron carriers, used in a great variety of life essential enzymes, and, in particular in my following example, nitrogenase, nitrogen-fixing enzymes. There is evidence, hard to be overlooked, of interdependence and irreducible structures in this synthesis pathway. A big number of proteins with the most varied functions are required, machines that build machines, a clearly applied manufacturing logic contributing for evident distant goals, and many of the tiny molecules, prosthetic groups, cofactors etc. are essential, that means if just one tiny prosthetic group is missing in the whole pathway, nothing goes, and everything breaks down. To exemplify this, I will start with an enzyme, which is indispensable and irreducible.  If just Pantothenate kinase is missing, all the subsequent synthesis steps are meaningless, and in the end, nitrogenase cofactor clusters cannot be synthesized, no nitrogen reduction and no complex life would exist on earth. One tiny protein amongst thousands, all life essential. It's not that if one part in a protein is missing, nothing goes, but if a tiny part of a whole factory like production process is missing, the whole process will produce no useful end product. 

Coenzyme A cofactors are essential for the existence of life on earth, and supposedly extant in the last universal common ancestor ( LUCA ). 

During the biosynthesis of Coenzyme A, the fourth step, after the action of 3  enzymes, namely:

Pantothenate kinase, 
Phosphopantothenoylcysteine synthetase, 
Phosphopantothenoylcysteine decarboxylase, 

produces an intermediate product, namely  4'-phosphopantetheine. For more details, see:

Synthesis of Coenzyme A
http://reasonandscience.catsboard.com/t2691-synthesis-of-coenzyme-a

Phosphopantetheine, also known as 4'-Phosphopantetheine (ppant), is an essential prosthetic group of peptidyl carrier proteins (PCP)  derived from Coenzyme A. Prosthetic groups act as cofactors, which are non-protein chemical compounds that are required for an enzyme's activity. 4'-phosphopantetheinyl transferase (PPTase) proteins transfer an above-described 4′-phosphopantetheinyl [size=12](ppant) , which has the form and acts like an arm, from coenzyme A (CoA) to an invariant serine amino acid in a peptidyl carrier protein (PCP) which[/size] are central, and play a critical role in nonribosomal peptide synthetase (NRPS)  enzymology.

Let's give a closer look at Nonribosomal peptide synthetase (NRPS).  These are macromolecular machines and amongst the world’s biggest enzymes. They belong to a family of microbial mega-enzymes that produce natural products like siderophores in a similar fashion like ribosomes, which grow polypeptide chains to make proteins. Some of these molecular machines contain over 16,000 amino acids !!! These are gigantic molecular assembly lines, and their domains orchestrate multiple and diverse chemical reactions in a highly coordinated manner. Another science paper describes them as among the largest and most complicated enzymes in nature. Each of their domains is responsible for catalyzing a distinct chemical transformation with a controlled timing to construct natural peptidyl products of remarkable chemical complexity. Their precision is staggering, and omitting one amino acid substrate results in a total breakdown of product formation, indicating that initiation of peptide synthesis cannot occur at internal modules of the mega-synthetase complexes. 

Their products, peptides similar to protein peptide chains, are biosynthesized with remarkable fidelity, typically as a single major product after hundreds of enzymatic reactions. The fidelity of nonribosomal peptide synthetases (NRPSs)  comes from what is called the “thiotemplate mechanism” or “assembly-line enzymology.” In fact, these biosynthetic enzymes are assembly lines on a molecular scale.  NRPSs are macromolecular machines with modular assembly-line logic, a complex catalytic cycle, moving parts and multiple active sites.   The immense size of the  NRPS polypeptides derives from their underlying division-of-labor organization. The emerging picture is one of remarkable dynamics and conformational programming in which the chemical states of individual catalytic domains are communicated to the others, configuring the modules for the next stage in the biosynthesis. When the construction process is over, the product is liberated from the assembly line and then undergoes further processing reactions to attain its final bioactive form.

The complication with  NRPS systems is not only that multiple catalytic sites, which may be located on the same or different polypeptides, work in series but also that the substrates are tethered to carrier proteins. These features result in a set of mechanistic issues unique to assembly line systems: how the catalytic domains communicate with the carrier proteins and how these interactions are regulated, the balance between enzyme-substrate and protein-protein interactions in programming the biosynthesis, how the multiple subunits recognize their correct partners and avoid incorrect associations are yet questions to be answered.

The so-called A domain is a protein subunit found in all  Nonribosomal peptide synthetase modules and is an essential catalytic component that also functions as a gatekeeper for the selection of amino acid building blocks during nonribosomal peptide biosynthesis.  The A domain selectively incorporates cognate amino acids into peptide-based natural products from a much larger monomer pool, including all 20 proteinogenic amino acids as well as a number of non-proteinogenic amino acids, aryl acids, fatty acids, and hydroxy acid building blocks. A domains are able to differentiate between a vast number of possible substrates - More than 300 amino acids, including D-configured, N-methylated, and other unusual non-proteinogenic residues, are found in the products.

Mispriming and the insertion of wrong peptides occur quite regularly, which interrupt the natural product assembly. Dead-ended and mis-primed PCP species are repaired by the action of so-called type II thioesterases (TEII). 

My comment: This is nothing short than amazing.  To illustrate this: Let us suppose that the genetic information has been encoded to produce a secondary metabolic product requiring a peptide chain of 10 substrates. The Nonribosomal peptide synthetase  (NRPS)  then requires 10 individual modules. Each has an A domain subunit, which is able to distinguish amongst 300 different products and to pick the right one to incorporate into the maturating peptide chain. They are like a hand and a glove. How they distinguish amongst 300+ substrates and work in a catalytically efficient manner is unknown.   That means the A domain of this complex production line is able to sort out amongst 300 substrates, is able to integrate them in the peptide chain, and if a wrong substrate is inserted,  type II thioesterases (TEII) repair the chain. In order to know what is the wrong product, you need to know what is the right one. How could evolution figure that out? 

Amongst many products, Nonribosomal peptide synthetases produce siderophores. 

Although iron is one of the most abundant elements on Earth, the environment is usually oxygenated, non-acidic, and aqueous. Under these conditions, extracellular iron is predominantly found in the poorly soluble ferric (oxydized Fe3+) state.  Many microorganisms, including some fungi, also secrete low molecular weight compounds known as siderophores into their surroundings, which form high-affinity (~10−33 M) complexes with ferric iron to make it bioavailable for uptake. Transporters on the cell surface then recapture the Fe3+-siderophores complexes.

Following are the required steps:

Iron uptake in Gram-negative bacteria involves four distinct steps: 

(i) siderophore synthesis,  (ii) siderophore secretion into the extracellular space,  (iii) iron chelation ( binding ) by the siderophores, and  (iv) siderophore/ iron uptake via complexes in the outer membrane and the intermembrane space as well as in the plasma membrane. 

as outlined above, non-ribosomal peptide synthetases are required and essential, because they produce the siderophores needed to chelate iron in the extracellular space. The third set of components is essential for the transfer of the iron-loaded siderophores through the outer membrane and periplasmic space into the cytoplasm. This process requires b-barrel shaped TonB-dependent transporters (TBDTs) in the outer membrane, an Exb/TonB complex localized in the plasma membrane, which regulates the TBDT, and siderophore uptake systems annotated as Fhu, Fec or Fut complexes

Maintaining adequate intracellular levels of transition metals is fundamental to the survival of all organisms. 47 While all transition metals are toxic at elevated intracellular concentrations, metals such as iron, zinc, copper, and manganese are essential to many cellular functions. In prokaryotes, the concerted action of a battery of membrane-embedded transport proteins controls a delicate balance between sufficient acquisition and overload.

It is estimated that 30–45% of known enzymes are metalloproteins that depend on a metal co-factor for their function.  Often, the co-factor is a transition metal such as iron, manganese, zinc, or copper. As a result, many essential physiological processes including respiration, photosynthesis, replication, transcription, translation, signal transduction, and cell division depend on the presence of transition metals. However, transition metals are toxic at elevated intracellular concentrations as they can perturb the cellular redox potential, produce highly reactive hydroxyl radicals k , and displace functionally important metal co-factors from their physiological locations.

Biosynthesis of the Cofactors of Nitrogenase  
http://reasonandscience.catsboard.com/t2429-biosynthesis-of-the-cofactors-of-nitrogenase

Fe–S clusters are among the most ancient types of prosthetic groups. The biosynthesis of the MoFe protein is extremely complex. FeS cluster assembly is a complex process involving the mobilization of Fe and S atoms from storage sources, their assembly into [Fe-S] form, their transport to specific cellular locations, and their transfer to recipient apoproteins. 30  Biological Fe-S cluster assembly is tightly regulated within cells.

Beside Iron, Molybdate, and sulfur enters by similarly complex import and regulation processes as for Iron import, and are processed to form  Mo-S containing species. Iron and sulfur are combined. Scaffold proteins assist the combination and activation, and finally, the ‘‘apo- MoFe protein.’’  is formed. This is, of course, a very short of the description of an extremely complex, not fully understood process.

The above-described process is life essential. ammonia is required to make amino acids. Nitrogenase enzymes are essential to fix nitrogen, and produce ammonia. Ammonia was not freely available on a prebiotic earth, as i described in a previous article:

Availability of ammonia in a prebiotic earth
http://reasonandscience.catsboard.com/t2689-availability-of-ammonia-in-a-prebiotic-earth

I think above shows, how robust the case for intelligent creation is, upon the scientific evidence and knowledge available today in regards of molecular processes. Life is designed. That's not an argument based on lack of understanding or gaps or ignorance, but it stands solid and firm on scientific ground, and positive evidence.  Cheers.

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The external thioesterase of the Surfactin-Synthetase
2010 Apr 14
The type II thioesterase (TEII), is a crucial repair enzyme for the regeneration of functional 4′-PP cofactors of holo T-domains of NRPS 15 Mispriming of 4′-PP cofactors by acetyl- and short chain acyl-residues interrupts the biosynthetic system. This repair reaction is very important, since roughly 80% of coenzyme A (CoA), the precursor of the 4′-phosphopantetheine cofactor, is acetylated in bacteria.

Non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) found in bacteria, utilize two different types of thioesterases  a for the production of highly active biological compounds. Type I thioesterases (TEI) catalyze the release step from the assembly line3 of the final product where it is transported from one reaction center to the next as a thioester linked to a 4′-phosphopantetheine cofactor (4′-PP) that is covalently attached to thiolation (T) domains4-9. The second enzyme involved in the synthesis of these secondary metabolites, the type II thioesterase (TEII), is a crucial repair enzyme for the regeneration of functional 4′-PP cofactors of holo T-domains of NRPS and PKS systems11-13. Mispriming of 4′-PP cofactors by acetyl- and short chain acyl-residues interrupts the biosynthetic system. This repair reaction is very important, since roughly 80% of coenzyme A (CoA), the precursor of the 4′-phosphopantetheine cofactor, is acetylated in bacteria14. Here we report the first three-dimensional structure of a type II thioesterase free and in complex with a T domain. Comparison with structures of TEI enzymes3, 15 shows the basis for substrate selectivity and the different modes of interaction of TEII and TEI enzymes with T domains. In addition, we show that the TEII enzyme exists in several conformations of which only one is selected upon interaction with its native substrate, a modified holo-T domain.


Iron Uptake and Homeostasis in Prokaryotic Microorganisms Non-ri10
Iron Uptake and Homeostasis in Prokaryotic Microorganisms Non-ri10
Iron Uptake and Homeostasis in Prokaryotic Microorganisms Non-ri11

Nearly 100 years ago, Henry Ford demonstrated the full strength of economist Adam Smith’s insights into productivity and the division of labour when he established the first moving assembly line. By shuttling partially constructed cars mechanically from one worker to the next, each performing a single specific task, Ford’s assembly line could issue a new Model T every three minutes. This manufacturing method provided the foundation of modern mass production. But nature employed much the same approach for constructing molecules long before humans existed to ponder questions of economy and efficiency. On page 824 of this issue, Walsh and colleagues1 identify a previously unrecognized link in one such biological assembly line — an enzyme that could some day be exploited by chemists to modify complex, naturally occurring compounds. The enzymes that form the polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) families are responsible for the biosynthesis of many useful compounds, including the antibiotics erythromycin and vancomycin, and the antitumour drug epothilone. These multi-subunit enzymes are the molecular equivalents of moving assembly lines: growing substrate molecules are handed, bucket-brigade style, from one specialized catalytic site to the next, with each site performing a specific and predictable function (Fig. 1). The PKS assembly line starts by recruiting The PKS assembly line starts by recruiting small building-blocks (such as acetate and propionate molecules, which contain ‘acyl’ chemical groups) onto carrier proteins. The building-blocks are then bonded together in reactions catalysed by a ‘ketosynthase’ region of the PKS. The resulting substrate may then be chemically tailored by various other enzyme domains, before being passed on to another ketosynthase for a further round of extension and modification. The cycle is repeated until the finished molecule is finally offloaded. The various catalytic domains may exist as discrete enzymes (as in type II PKS), or be connected end to end, like beads on a string (as in type I PKS), but in both cases the biosynthetic strategy remains the same. The NRPS cycle is very similar to that of PKS enzymes, except that it uses amino acids as building-blocks. Thus, amino acids become bound to peptidyl carrier proteins (PCPs); PCP-bound amino acids are joined together with amide bonds to form peptides, in catalytic sites known as condensation domains; tailoring regions may then modify the newly formed peptide before passing it along for further cycles of extension and tailoring; and finally, the finished product is cleaved from the enzyme. The PKS and NRPS enzymes each produce very different products, but the logic they use is strikingly similar — so similar, in fact, that they can easily cooperate to construct hybrid PKS–NRPS products such as epothilone. 

Atlas of nonribosomal peptide and polyketide biosynthetic pathways reveals common occurrence of nonmodular enzymes
This study demonstrates the widespread distribution of nonribosomal peptide synthetase and modular polyketide synthase biosynthetic pathways across the three domains of life, by cataloging a total of 3,339 gene clusters from 2,699 genomes. Our analysis suggests that noncanonical nonmodular biosynthetic enzymes are common in bacteria. Proteobacteria, Actinobacteria, Firmicutes, and Cyanobacteria in bacteria and Ascomycota in fungi contained higher number of these gene clusters and are likely to produce a wide variety of nonribosomal peptide and polyketide types of natural products.

Nonribosomal peptide synthesis versus ribosomal peptide/protein synthesis
The activation of the amino acid substrate is similar in both biosynthetic systems, but the enzymes involved, aa-tRNA synthetase in ribosomal and adenylation domains in nonribosomal synthesis, are structurally and catalytically unrelated. The activated amino acid is loaded to the transport unit, tRNA in ribosomal and the thiolation domain (peptidyl carrier protein, PCP) in nonribosomal synthesis. Both the ribosomal and nonribosomal machinery have properties that make them well-suited for their respective functions. Because of the need for precision in primary metabolism, ribosomal peptide synthesis involves several proofreading mechanisms that are
absent from the nonribosomal system. However, ribosomal peptide synthesis is normally restricted to a set of 20 amino acids as building blocks for proteins, whereas several hundred substrates of NRPSs are known to date. Thus, structural diversity is a predominant feature of nonribosomally produced peptides.




Iron Uptake and Homeostasis in Prokaryotic Microorganisms Non-ri12

Modular structure of nonribosomal peptide synthetases
In ribosomal and nonribosomal peptide synthesis, the activation of amino acids is followed by peptide bond formation, and the steps are repeated until the final length of the peptide is attained. In NRPSs this is achieved through a modular structure, with one module for each amino acid to be incorporated into the peptide.  The minimal module required for the addition of an amino acid to the growing peptide (Figure 3) consists of a condensation (C), an adenylation (A) and a thiolation (T) domain. The A-domain recognizes the amino acid substrate and activates it, first through the formation of an aminoacyl adenylate and then via covalent binding of the activated amino acid as a thioester to the phosphopantetheine group of the T-domain. The C-domain catalyses the formation of a peptide bond between the aminoacyl or peptidyl moiety and the free amino group of the downstream aminoacyl moiety. In addition to these three core domains, modules can contain several alternative domains that introduce a modification of (epimerization domain), N-methylation (N-methyltransferase -amino group, or the heterocyclization of Ser, Thr or Cys residues (heterocyclization domain). The last step in nonribosomal peptide biosynthesis involves release of the assembled peptide from the NRPS into solution. In the majority of cases, this is accomplished by an extra domain located at the C-terminus of the last module of the NRPS, which can be a thioesterase (TE) domain, a variant of a C domain or a reductase domain. In silico recognition of individual domains on the protein level can be achieved using domain-specific highly conserved sequence motifs. In many NRPS gene clusters the order and number of modules is co-linear with the amino acid sequence and length of the peptide. Consequently, it is possible by analysing the sequence of NRPS genes to elucidate the composition of the peptide, provided that the substrate specificities of the adenylation domains are known or can be deduced.

Iron Uptake and Homeostasis in Prokaryotic Microorganisms Non-ri13


Substrate recognition by nonribosomal peptide synthetases

A substantial variety of siderophore structures are produced from similar NRPS assembly lines. An NRPS assembly line is comprised of autonomously folding domains, bundled together into functional modules to carry out steps of monomer selection and activation, chain elongation, and then chain termination. Each NRPS assembly line needs to be organized to carry out four kinds of catalytic operations. First, each of the PCP domains must be converted from the apo form to the holo form, bearing the phosphopantetheinyl arm

Iron Uptake and Homeostasis in Prokaryotic Microorganisms Sidero10
Figure 3. Initiation of siderophore synthesis. 
(A) PPTase priming equation on apo PCP; 
(B) two-step A domain equation; 
(C) C domain equation.





Humans also use an acidic environment to facilitate uptake of dietary iron. Uptake mainly occurs though enterocytes in the duodenum, which receives the acidic contents of the stomach. Iron can then be absorbed for storage in intestinal cells or delivery to other cells

In mammals, iron enters the cell through a variety of transport systems, including the endosomal transporter DMT1

Iron Uptake and Homeostasis in Prokaryotic Microorganisms Fphar-10

Distribution of iron in the cytosol of mammalian cells. Iron enters the cytosolic labile iron pool (LIP) as Fe(II) and may be coordinated by reduced glutathione (GSH). Most of the iron entering the LIP is delivered to mitochondria for the synthesis of heme and Fe–S clusters. In the cytosol, homodimers of Grx3 or heterooligomers of Grx3 and BolA2 coordinate [2Fe-2S] clusters with the help of GSH. These clusters may be used for the metallation of cytosolic Fe-S enzymes (via the cytosolic iron–sulfur cluster assembly machinery, CIA) and may have a role in mitochondrial iron delivery in erythroid tissues. PCBP1 and PCBP2 bind iron in the cytosol and deliver it to target non-heme iron enzymes, such as ferritin, 2-oxoglutarate-dependent dioxygenases (mononuclear iron enzymes such as PHD2 or FiH1), and oxo- or peroxo-bridged diiron oxygenases (dinuclear iron enzymes such as DOHH). The source of iron for PCBPs is not known, but could include components of the LIP coordinated by GSH or iron coordinated by Grx3. PCBP2 may directly acquire iron from transporters such as DMT1 and may also deliver iron to ferroportin for efflux. Some intracellular organelles have been omitted for clarity. Red spheres indicate inorganic sulfide as part of an Fe–S cluster.

Composition of the Cytosolic Labile Iron Pool
From a conceptual standpoint, the cytosol must contain a kinetically labile, metabolically accessible pool of iron so that iron can be distributed to its various sites of utilization, transport, or storage. The iron initially entering the cytosol is also in the reduced state.

Proteins Involved in Cytosolic Iron Distribution: Monothiol Glutaredoxins
Candidates for physiologic Fe(II) ligands include citrate, cysteine, and reduced glutathione (GSH). Analysis of these potential ligands in vitro suggests that Fe–GSH complexes are likely the only species that forms at significant levels in cells, largely because GSH is present at relatively high (2–10 mM) levels in the cytosol and exhibits moderate affinity (Kd∼8 μM) for Fe(II).  Fe–GSH complexes  have a critical role in maintaining iron distribution and homeostasis in eukaryotic cells. In addition to its role as a ligand in the cytosolic LIP, GSH coordinates iron as part of the Fe–S carrier complex formed by cytosolic monothiol glutaredoxins (reviewed in Glutaredoxins: roles in iron homeostasis). The glutaredoxins are a ubiquitous class of enzyme with a thioredoxin fold and are generally thought to function as thiol-disulfide oxidoreductases with roles in Glutathione conjugation. 

The term metallochaperone has been used to describe proteins that directly deliver metals to target enzymes or transporters through metal-mediated protein–protein interactions. Iron chaperone activity has been demonstrated for the poly C binding protein (PCBP) family of proteins. PCBP1 was initially identified as an iron chaperone for ferritin, the ubiquitous iron storage protein. Ferritin, composed of 24 subunits of H- and L-isoforms, functions as a cellular repository of surplus iron by accommodating up to 4500 iron atoms within its spherical core. Delivery of iron atoms into the hollow sphere of ferritin occurs via the hydrophilic channels formed by the carboxylate side chains along the threefold symmetry axes in the heteropolymer. Initially, ferritin accrues iron atoms in the ferrous form, which are then oxidized to ferric iron by the ferroxidase center of H-ferritin, located in the interior of the ferritin cavity. Even though the regulatory mechanisms of this gene product have been extensively studied under various physiological conditions, the cytosolic trafficking system directing iron to the metalloprotein had been elusive until the recent discovery of the iron chaperone activity of PCBP1.

Uptake of Ferrous Iron 7
For almost all microorganisms iron is an essential element involved in many important reactions involving, among others, [Fe-S] proteins and haem in cytochromes. Under normal environmental conditions, iron presents two oxidation states, Fe2 and Fe3, that are particularly suitable in oxido-reduction reactions. While Fe2 is the dominant form under anaerobic conditions, Fe3  is the major form in oxygenated environments. This presents a problem for microorganisms with an aerobic lifestyle because of the extremely low solubility of the ferric iron.  

Siderophores are strong iron chelators, secreted by many organisms, including bacteria, fungi, yeast and monocotyledonous plants to solubilize, bind and make available iron in the environment. Generally, organisms synthesize and secrete these low molecular weight chelators to bind Fe(III) and then transport the ferri-siderophore complex through the cell membrane. Unlike other organisms,

Gram-negative bacteria possess an outer membrane (OM) as well as a cytoplasmic membrane (CM), which presents an additional barrier to the exchange of solutes. As ferri-siderophores are too large to passively diffuse through the OM porins, they must be actively transported across the membrane by specific receptor proteins  The OM receptors/transporters bind the ferri-siderophore complexes and directly interact with the energizing TonB-ExbB-ExbD complex in the inner membrane to allow the iron complex to be transported into the periplasmic space. This transport process involves three components: 

(i) OM localized transporters; 
(ii) a CM-localized TonB-ExbB-ExbD complex, and 
(iii) ion electrochemical potential

Over the past three decades, many aspects of this TonB-ExbB-ExbD-dependent transport system have been revealed. The crystal structures of several OM transporters and their complexes with TonB are now known, the signal transduction of OM transporters by interaction with TonB has been elucidated  and the rotational mechanism of TonB motion has been reported . However, with regard to the substrates of the transport system, we are probably only seeing the ‘tip of the iceberg'. Originally, iron complexes and vitamin B12 were thought to be the main substrates of the TonB-ExbB-ExbD system, but more and more new substrates have been found to be transported, including citrate, transferrin, hemoproteins, heme, phages, colicins, maltodextrins, nickel chelators and sucrose.

TonB-dependent transporters: regulation, structure, and function

TonB-dependent transporters (TBDTs) are bacterial outer membrane proteins that bind and transport ferric chelates called siderophores, as well as vitamin B12, nickel complexes, and carbohydrates. The transport process requires energy in the form of protonmotive force and a complex of three inner membrane proteins, TonB-ExbB-ExbD, to transduce this energy to the outer membrane. The siderophore substrates range in complexity from simple small molecules such as citrate to large proteins like serum transferrin and haemoglobin. Because iron uptake is vital for almost all bacteria, expression of TBDTs is regulated in a number of ways that include metal-dependent regulators, σ/anti-σ factor systems, small RNAs, and even a riboswitch. In recent years many new structures of TBDTs have been solved in various states, resulting in a more complete picture of siderophore selectivity and binding, signal transduction across the outer membrane, and interaction with TonB-ExbB-ExbD. However, the transport mechanism is still unclear. In this review, we summarize recent progress in understanding regulation, structure and function in TBDTs and questions remaining to be answered.

Transport into Gram-negative organisms is initiated by passage of the transported species across the outer membrane and into the periplasmic space prior to inner membrane translocation. The uptake of iron is particularly important for bacterial growth and synthesis of outer membrane iron transporters (called TonB-dependent transporters, TBDTs) is therefore regulated in a variety of ways. While iron complexes constitute the majority of substrates for TBDTs, vitamin B12, nickel chelates, and carbohydrates are also transported by this mechanism. These transporters show high affinity and specificity for metal chelates called siderophores and require energy derived from the protonmotive force across the inner membrane to transport them. To tap this energy source, TBDTs must interact with an inner membrane protein complex consisting of TonB, ExbB, and ExbD.

The first crystal structures of two Escherichia coli TonB-dependent transporters, ferrichrome transporter (FhuA) and ferric enterobactin transporter (FepA), showed that TBDTs use a 22-stranded β-barrel to span the outer membrane with an unanticipated ‘plug’ domain folded into the barrel interior. The plug domain functions to bind a specific metal chelate at the extracellular side of the membrane and to interact with TonB-ExbB-ExbD at the periplasmic side of the outer membrane. In these ‘ground state’ structures, the plug domain completely occludes the barrel pore, revealing an unexpected complexity for siderophore transport. There has been significant recent progress in structure determination of TBDTs, with a total of 45 structures solved to date, representing 12 unique transporters. In this review, we summarize new data on the complex regulation of TBDTs, structural similarities and differences, and new functional data pertaining to the transport mechanism. We will focus on E. coli, but include information on other Gram negative bacteria where appropriate.

Iron Uptake and Homeostasis in Prokaryotic Microorganisms Tonb-e11

Transport and regulation of siderophores
Transport of ferric siderophores across the outer membrane derives energy from the inner membrane protonmotive force. This requires an energy-transducing TonB complex in the inner membrane (blue), consisting of TonB, ExbB and ExbD proteins. TonB interacts with outer membrane transporters (TBDT) at the TonB-box motif. Transport of ferric siderophores across the inner membrane requires a periplasmic binding protein and an ABC transporter. Once the ferric siderophore enters the cytoplasm, ferric ion (Fe3+) is reduced to ferrous ion (Fe2+), which is destined for storage or incorporation into enzymes. Excess Fe2+ (which could induce the formation of radicals harmful to the cell) binds to the repressor protein Fur, which in turn binds target promoters (Pfur) and inhibits transcription of siderophore transport genes. Some TBDTs, such as E. coli FecA are additionally regulated by σ/anti-σ factor systems. In addition to transporting diferric dicitrate, FecA regulates the expression of fecABCDE transport genes initiated by the binding of ferric citrate to FecA. This involves the N-terminal extension of FecA (green), the inner membrane regulator FecR (σ regulator, pink), and the cytoplasmic sigma factor FecI (ECF σ factor, pink). Both transport and induction require energy transduction from the TonB-ExbB-ExbD complex in the inner membrane.


Iron Uptake and Homeostasis in Prokaryotic Microorganisms The_st10

The structure of the (prototype) TBDT FhuA
TBDTs have an N-terminal plug domain that sits inside a C-terminal 22-stranded β-barrel domain. The conserved TonB box is found near the N-terminus of the plug domain facing the periplasm and is generally thought to remain sequestered inside the β-barrel domain in the absence of ligand. Upon binding ligand, a conformational change leads to exposure of the TonB box and subsequent interaction with TonB and siderophore transport. Panel a represents the FhuA-ferrichrome crystal structure (1BY5) with FhuA shown in ribbon and ferrichrome in spacefill model, panel b represents only the beta-barrel domain, panel c represents only the plug domain, and panel d shows the FhuA apo structure (1BY3) with those residues with at least 50% conservation highlighted in blue. Top view represents the extracellular view, side view represents the membrane view, and bottom represents the periplasmic view. The TonB box was found disordered in both structures and is represented by dashed lines.


Structure and Function of TBDTs
An analysis of the original four TBDTs showed that all of them have the same domain architecture: a 22-stranded transmembrane β-barrel encloses a globular plug domain (Figure 2). Ligand binding sites are formed from residues on the extracellular side of the plug domain, as well as from residues on the walls and extracellular loops of the β-barrel. The TonB box is found at the N-terminus of the plug domain, and in some structures protrudes into the periplasm. In others, the TonB box is tucked up into the plug domain within the barrel or is disordered and not visible in the structures. A structure-based sequence alignment revealed conserved motifs in the plug and barrel which are close to one another and interact. Finally, an analysis of water molecules located at the plug-barrel interface revealed that the plug is highly solvated, resembling a transient protein complex and suggesting conformational change and/or movement of the plug within the barrel during transport. In the following sections, we will outline some of the significant structural and functional studies done with TBDTs in recent years.

Structural insight into the role of the Ton complex in energy transduction

Energy-coupled transport and signal transduction through the Gram-negative outer membrane via TonB-ExbB-ExbD-dependent receptor proteins

Iron Uptake and Homeostasis in Prokaryotic Microorganisms Tonb-e10


The FeoABC System
The ferrous iron Fe2 is highly soluble compared to the oxidized Fe3 and is stable under anaerobic conditions and at low pH.5 Soluble Fe2 is transported into cells via a transport system termed Feo, composed of a permease FeoB and the proteins FeoA and FeoC. This system is probably ancient since the primitive atmosphere of Earth was anoxic, resulting in the high abundance of this form of iron.  Feo genes are present in more than 1000 bacterial genomes.     The major component is the FeoB permease, which is a polytopic cytoplasmic membrane protein with eight transmembrane domains in the majority of cases (Figure 11.1B).

Iron Uptake and Homeostasis in Prokaryotic Microorganisms Feoabc10

The protein has an N-terminal domain in the cytoplasm with a GTPase function, which is typical of eukaryotic G-proteins such as Ras.  It possesses low affinity for GTP coupled with a low hydrolysis rate but the release rate of GDP is fast. The N-terminal FeoB domain forms a trimeric structure where the G-domain covers amino acids 1–170 while the second part of the FeoB N-domain, termed the S-domain, could act as a gate for Fe2+. FeoA has an SH3 domain typical of GTPase activating proteins that interact with eukaryotic G-proteins. The C-terminal membrane domain of FeoB has two ‘‘gates’’ in opposite orientations, similar to those in the iron permease of yeast, Ftr1p.12 There are also several conserved cysteine residues in transmembrane domains 3 and 6 and in the first cytoplasmic loop (Figure 11.1B). It has been proposed that the conserved Cys residues in the gates 1 and 2 transmembrane domains serve as ligands for Fe2+.

The small FeoC protein is predicted to contain an Fe-S cluster and it presents a LysR-regulator-like winged-helix motif at its N-terminus, suggesting that it could be involved in the regulation of the transcription of the feo operon. Recently, it was demonstrated that FeoC also interacts with the N-terminal cytoplasmic domain of FeoB, suggesting a role in the modulation of Fe2 uptake. Although the feoC gene is found only in g-Proteobacteria, other bacteria encode other small Fe-S cluster proteins in their feo operons. A role for the FeoA protein has been proposed recently based on the fact that uptake of Fe21 is reduced in a feoA mutant in which expression of the FeoB protein is unaffected. The same study demonstrated an interaction between FeoA and FeoB via a bacterial two-hybrid system. Recently, the structure of FeoA has been solved and the results do not support the hypothesis that FeoA assists FeoB in GTP hydrolysis. Rather they suggest a direct interaction between FeoA and the so-called ‘‘core’’ membrane domain of FeoB.

The advent of oxygen was a catastrophic event for most living organisms, and can be considered to be the first general irreversible pollution of the earth. The oxidation of iron resulted in the loss of its bioavailability as Fe(II) was replaced by insoluble Fe(III) 9 A new iron biochemistry became possible after the advent of oxygen, with the development of chelators of Fe(III), which rendered iron once again accessible, and with the control of the potential toxicity of iron by its storage in a water soluble, non-toxic, bio-available storage protein (ferritin). Biology also discovered that whereas enzymes involved in anaerobic metabolism were designed to operate in the lower portion of the redox spectrum (attaining values of close to þ0.6 V for iron itself), the arrival of dioxygen created the need for a new redox active metal which could attain higher redox potentials

This raises the question when Siderophores emerged. Siderophores are amongst the strongest soluble Fe3+ binding agents known. They had no business if FeIII was not existent prior the great oxygenation event.



The amazing molecular assembly lines of multidomain non-ribosomal peptide synthetases (NRPSs) 

http://reasonandscience.heavenforum.org/t2443-iron-uptake-and-homeostasis-in-prokaryotic-microorganisms#5213

If you thought there is only one way to make proteins ( polypeptide amino-acid molecules )  by the well-known process  DNA => RNA polymerase => mRNA => Ribosome + tRNA =>>  amino-acid polypeptides, you did not hear until now about NRPS or nonribosomal peptide synthetases.

In the book A privileged Planet, and at page 201,  Gonzales writes: 
The strong nuclear force is responsible for holding protons and neutrons together in the nuclei of atoms. In such close quarters, it is strong enough to overcome the electromagnetic force and bind the otherwise repulsive, positively charged protons together. It is as short-range as it is strong, extending no farther than atomic nuclei. But despite its short range, changing the strong nuclear force would have many wide-ranging consequences, most of them detrimental to life.  The periodic table of the elements would look different with a changed strong nuclear force. If it were weaker, there would be fewer stable chemical elements. The more complex organisms require about twenty-seven chemical elements, iodine being the heaviest (with an atomic number of 53). Instead of ninety-two naturally occurring elements, a universe with a strong force weaker by 50 percent would have contained only about twenty to thirty. This would eliminate the life-essential elements iron and molybdenum. 

Molybdenum is life essential. It is required in nitrogenase enzymes. Following the life-essential elements :

Essential elements and building blocks for the origin of life
http://reasonandscience.heavenforum.org/t2437-essential-elements-and-building-blocks-for-the-origin-of-life

Nitrogenase enzymes work like molecular sledge-hammer breaking the molecular triple bond of nitrogen, and transform nitrogen gas into ammonia, essential for the makeup of living things, and it uses in its active site as co-factor molybdenum.

The Nitrogenase enzyme,  the molecular sledgehammer  
http://reasonandscience.heavenforum.org/t1585-nitrogenase#2406

With assistance from an energy source (ATP) and a powerful and specific complementary reducing agent (ferredoxin), nitrogen molecules are bound and cleaved with surgical precision. In this way, a ‘molecular sledgehammer’ is applied to the NN bond, and a single nitrogen molecule yields two molecules of ammonia. The ammonia then ascends the ‘food chain’, and is used as amino groups in protein synthesis for plants and animals. This is a very tiny mechanic , but multiplied on a large scale it is of critical importance in allowing plant growth and food production on our planet to continue.

How are the MOLYBDENUM COFACTORs, or the essential active sites for nitrogenase function that contains molybdenum, synthesized?

Molybdenum, essential for life
http://reasonandscience.heavenforum.org/t2430-molybdenum-essential-for-life

For the starting point of molybdenum co-factor maturation, Iron - Sulfur ( FE/S) clusters are used. 

Biosynthesis of Iron-sulfur clusters, basic building blocks for life  
http://reasonandscience.heavenforum.org/t2285-iron-sulfur-clusters-basic-building-blocks-for-life

In order to make FE/S clusters, the cell needs the uptake of Iron and Sulfur. How does that happen in the cell ? 

Sulfur essential for life
http://reasonandscience.heavenforum.org/t2433-sulfur-essential-for-life

Iron Uptake and Homeostasis in Cells
http://reasonandscience.heavenforum.org/t2443-iron-uptake-and-homeostasis-in-prokaryotic-microorganisms

Iron Bioavailability
Although iron is one of the most abundant elements on Earth, the environment is usually oxygenated, non-acidic, and aqueous. Under these conditions, extracellular iron is predominantly found in the poorly soluble ferric (Fe3+) state. One way that organisms such as yeast improve iron bioavailability is by acidifying the local environment.  By lowering the pH of the surrounding environment, organisms facilitate solubilization and uptake of iron. ATP-driven proton transporters move H+ ions from the cytosol across the plasma membrane to control the pH at the cell surface.

Question: Had this system not have to emerge fully developed right from the beginning in order to facilitate and make Iron uptake into the cell even possible?

Uptake of Iron by micro-organisms like Bacteria and fungi
Many microorganisms, including some fungi, also secrete low molecular weight compounds known as siderophores into their surroundings, which form high-affinity (~10−33 M) complexes with ferric iron to make it bioavailable for uptake. Transporters on the cell surface then recapture the Fe3+-siderophores complexes.  

The fascinating part: To make these siderophores, amazing assembly lines in the cell exist. 

Enzymes line up for assembly 1 ,  how non-ribosomal peptide synthetase (NRPS) work :

Nearly 100 years ago, Henry Ford demonstrated the full strength of economist Adam Smith’s insights into productivity and the division of labor when he established the first moving assembly line. By shuttling partially constructed cars mechanically from one worker to the next, each performing a single specific task, Ford’s assembly line could issue a new Model T every three minutes. This manufacturing method provided the foundation of modern mass production. But nature employed much the same approach for constructing molecules long before humans existed to ponder questions of economy and efficiency.  Walsh and colleagues identify a previously unrecognized link in one such biological assembly line — an enzyme that could someday be exploited by chemists to modify complex, naturally occurring compounds. The enzymes that form the polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) families are responsible for the biosynthesis of many useful compounds, including the antitumor drug epothilone. These multi-subunit enzymes are the molecular equivalents of moving assembly lines: growing substrate molecules are handed, bucket-brigade style, from one specialized catalytic site to the next, with each site performing a specific and predictable function. The PKS assembly line  starts by recruiting small building-blocks onto carrier proteins. The building blocks are then bonded together in reactions catalysed by a ‘ketosynthase’ region of the PKS. The resulting substrate may then be chemically tailored by various other enzyme domains, before being passed on to another ketosynthase for a further round of extension and modification. The cycle is repeated until the finished molecule is finally offloaded. The various catalytic domains may exist as discrete enzymes (as in type II PKS), or be connected end to end, like beads on a string (as in type I PKS), but in both cases the biosynthetic strategy remains the same. The NRPS cycle is very similar to that of PKS enzymes, except that it uses amino acids as building-blocks. Thus, amino acids become bound to peptidyl carrier proteins (PCPs); PCP-bound amino acids are joined together with amide bonds to form peptides, in catalytic sites known as condensation domains; tailoring regions may then modify the newly formed peptide before passing it along for further cycles of extension and tailoring; and finally, the finished product is cleaved from the enzyme. The PKS and NRPS enzymes each produce very different products, but the logic they use is strikingly similar — so similar, in fact, that they can easily cooperate to construct hybrid PKS–NRPS products such as epothilone.

This assembly line, together with non-ribosomal codes, produces siderophores, essential for the uptake of iron in bacterias. Iron, essential for the synthesis of FE/S clusters. FE/S clusters, essential for the formation of Molybdenum cofactors.  All above-described cell processes must exist prior life began, in order to produce Molybden co-factors.

Minerals containing molybdenum are key in assembling atoms into life-forming molecules. The researcher points out that boron minerals help carbohydrate rings to form from pre-biotic chemicals, and then molybdenum takes that intermediate molecule and rearranges it to form ribose, and hence RNA. Chromium, molybdenum, selenium, and vanadium, for example, are essential for building proteins, and proteins serve as life’s molecular “factories.”

All this brings us to the conclusion, that the scientific evidence exposed points to the requirement of all these processes to emerge all at once,  intelligently created, fully set up, and working right from the beginning. A stepwise, gradual origin of these processes is impossible.  

1. https://sci-hub.bz/http://www.nature.com/nature/journal/v448/n7155/full/448755a.html

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