https://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)
(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.
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.
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
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
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:
(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).
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.
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.
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.
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
https://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
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
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.
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
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).
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.
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.
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)
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
An acyl group is a moiety derived by the removal of one or more hydroxyl groups from an oxoacid 21
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.
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
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.
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
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