The Structure of a Transient Complex of a Nonribosomal Peptide Synthetase and a Cytochrome P450 Monooxygenase

Studying the interplay between nonribosomal peptide synthetases (NRPS), a major source of secondary metabolites, and crucial external modifying enzymes is a challenging task since the interactions involved are often transient in nature. By applying a range of synthetic inhibitor‐type compounds, a stabilized complex appropriate for structural analysis was generated for such a tailoring enzyme and an NRPS domain. The complex studied comprises an NRPS peptidyl carrier protein (PCP) domain bound to the Cytochrome P450 enzyme that is crucial for the provision of β‐hydroxylated amino acid precursors in the biosynthesis of the cyclic depsipeptide skyllamycin. The structure reveals that complex formation is governed by hydrophobic interactions, the presence of which can be controlled through minor alterations in PCP structure that enable selectivity amongst multiple highly similar PCP domains.     

Formylation domain: an essential modifying enzyme for the nonribosomal biosynthesis of linear gramicidin

Formylation is an important part of ribosomal peptide synthesis of prokaryotes. In nonribosomal peptide synthesis, however, N-formylation is rather unusual and therefore so far unexplored. In this work, the first module of the linear gramicidin nonribosomal peptide synthetase, LgrA1, consisting of a hypothetical formylation domain, an adenylation, and a peptidyl carrier protein domain was tested for formyltransferase activity in vitro. We demonstrate here that the putative formylation domain does indeed transfer the formyl group of formyltetrahydrofolate (fH4F) onto the first amino acid valine using both cofactors N10- and N5-fH4F, respectively. Most important, the necessity of the formylated starter unit formyl-valine for the initiation of the gramicidin biosynthesis was tested by elongation assays with the bimodular system from LgrA. By omitting the formyl group donor, no condensation product of valine with the subsequent building block glycine was detected, whereas the dipeptide formyl-valyl-glycine was found when assayed in the presence of either formyl donor. The proven formylation activity of the first domain of LgrA represents a novel tailoring enzyme in nonribosomal peptide synthesis.     

Nine enzymes are required for assembly of the pacidamycin group of peptidyl nucleoside antibiotics

Pacidamycins are a family of uridyl peptide antibiotics that inhibit the translocase MraY, an essential enzyme in bacterial cell wall biosynthesis that to date has not been clinically targeted. The pacidamycin structural skeleton contains a doubly inverted peptidyl chain with a β-peptide and a ureido linkage as well as a 3'-deoxyuridine nucleoside attached to DABA(3) of the peptidyl chain via an enamide linkage. Although the biosynthetic gene cluster for pacidamycins was identified recently, the assembly line of this group of peptidyl nucleoside antibiotics remained poorly understood because of the highly dissociated nature of the encoded nonribosomal peptide synthetase (NRPS) domains and modules. This work has identified a minimum set of enzymes needed for generation of the pacidamycin scaffold from amino acid and nucleoside monomers, highlighting a freestanding thiolation (T) domain (PacH) as a key carrier component in the peptidyl chain assembly as well as a freestanding condensation (C) domain (PacI) catalyzing the release of the assembled peptide by a nucleoside moiety. On the basis of the substrate promiscuity of this enzymatic assembly line, several pacidamycin analogues were produced using in vitro total biosynthesis.     

Peptide macrocyclization: the reductase of the nostocyclopeptide synthetase triggers the self-assembly of a macrocyclic imine

Many biologically active natural products have macrocyclic structures. In nonribosomal peptides macrocyclization is commonly achieved via the formation of intramolecular ester or amide bond catalyzed by thioesterase domains during biosynthesis. A unique and so far unknown type of peptide cyclization occurs in the nostocyclopeptide, a macrocyclic imine produced by the terrestrial cyanobacterium Nostoc sp. ATCC53789. In this work we show that a C-terminal reductase domain of the nostocyclopeptide nonribosomal peptide synthetase catalyzes the reductive release of a linear peptide aldehyde and thereby triggers the spontaneous formation of a stable imino head-to-tail linkage. This type of molecular self-assembly induced by the reductive release of reactive aldehydes may be more commonplace in other complex nonribosomal peptides than originally thought.     

New Structural Data Reveal the Motion of Carrier Proteins in Nonribosomal Peptide Synthesis

The nonribosomal peptide synthetases (NRPSs) are one of the most promising resources for the production of new bioactive molecules. The mechanism of NRPS catalysis is based around sequential catalytic domains: these are organized into modules, where each module selects, modifies, and incorporates an amino acid into the growing peptide. The intermediates formed during NRPS catalysis are delivered between enzyme centers by peptidyl carrier protein (PCP) domains, which makes PCP interactions and movements crucial to NRPS mechanism. PCP movement has been linked to the domain alternation cycle of adenylation (A) domains, and recent complete NRPS module structures provide support for this hypothesis. However, it appears as though the A domain alternation alone is insufficient to account for the complete NRPS catalytic cycle and that the loaded state of the PCP must also play a role in choreographing catalysis in these complex and fascinating molecular machines.     

Diketopiperazine Formation in Fungi Requires Dedicated Cyclization and Thiolation Domains

Cyclization of linear dipeptidyl precursors derived from nonribosomal peptide synthetases (NRPSs) into 2,5‐diketopiperazines (DKPs) is a crucial step in the biosynthesis of a large number of bioactive natural products. However, the mechanism of DKP formation in fungi has remained unclear, despite extensive studies of their biosyntheses. Here we show that DKP formation en route to the fungal virulence factor gliotoxin requires a seemingly extraneous couplet of condensation (C) and thiolation (T) domains in the NRPS GliP. In vivo truncation of GliP to remove the CT couplet or just the T domain abrogated production of gliotoxin and all other gli pathway metabolites. Point mutation of conserved active sites in the C and T domains diminished cyclization activity of GliP in vitro and abolished gliotoxin biosynthesis in vivo. Verified NRPSs of other fungal DKPs terminate with similar CT domain couplets, suggesting a conserved strategy for DKP biosynthesis by fungal NRPSs.     

Assembly line termination in cylindrocyclophane biosynthesis: discovery of an editing type II thioesterase domain in a type I polyketide synthase

The termination step is an important source of structural diversity in polyketide biosynthesis. Most type I polyketide synthase (PKS) assembly lines are terminated by a thioesterase (TE) domain located at the C-terminus of the final module, while other PKS assembly lines lack a terminal TE domain and are instead terminated by a separate enzyme in trans. In cylindrocyclophane biosynthesis, the type I modular PKS assembly line is terminated by a freestanding type III PKS (CylI). Unexpectedly, the final module of the type I PKS (CylH) also possesses a C-terminal TE domain. Unlike typical type I PKSs, the CylH TE domain does not influence assembly line termination by CylI in vitro. Instead, this domain phylogenetically resembles a type II TE and possesses activity consistent with an editing function. This finding may shed light on the evolution of unusual PKS termination logic. In addition, the presence of related type II TE domains in many cryptic type I PKS and nonribosomal peptide synthetase (NRPS) assembly lines has implications for pathway annotation, product prediction, and engineering.     

Assembly line enzymology by multimodular nonribosomal peptide synthetases: the thioesterase domain of E. coli EntF catalyzes both elongation and cyclolactonization.

BACKGROUND: EntF is a 142 kDa four domain (condensation-adenylation-peptidyl carrier protein-thioesterase) nonribosomal peptide synthetase (NRPS) enzyme that assembles the Escherichia coli N-acyl-serine trilactone siderophore enterobactin from serine, dihydroxybenzoate (DHB) and ATP with three other enzymes (EntB, EntD and EntE). To assess how EntF forms three ester linkages and cyclotrimerizes the covalent acyl enzyme DHB-Ser-S-PCP (peptidyl carrier protein) intermediate, we mutated residues of the proposed catalytic Ser-His-Asp triad of the thioesterase (TE) domain. RESULTS: The Ser1138-->Cys mutant (kcat decreased 1000-fold compared with wild-type EntF) releases both enterobactin (75%) and linear (DHB-Ser)2 dimer (25%) as products. The His 1271-->Ala mutant (kcat decreased 10,000-fold compared with wild-type EntF) releases only enterobactin, but accumulates both DHB-Ser-O-TE and (DHB-Ser)2-O-TE acyl enzyme intermediates. Electrospray ionization and Fourier transform mass spectrometry of proteolytic digests were used to analyze the intermediates. CONCLUSIONS: These results establish that the TE domain of EntF is both a cyclotrimerizing lactone synthetase and an elongation catalyst for ester-bond formation between covalently tethered DHB-Ser moieties, a new function for chain-termination TE domains found at the carboxyl termini of multimodular NRPSs and polyketide synthases.     

Molecular analysis of the kirromycin biosynthetic gene cluster revealed beta-alanine as precursor of the pyridone moiety

Kirromycin is a complex linear polyketide that acts as a protein biosynthesis inhibitor by binding to the bacterial elongation factor Tu. The kirromycin biosynthetic gene cluster was isolated from the producer, Streptomyces collinus Tü 365, and confirmed by targeted disruption of essential biosynthesis genes. Kirromycin is synthesized by a large hybrid polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) encoded by the genes kirAI-kirAVI. This complex involves some very unusual features, including the absence of internal acyltransferase (AT) domains in KirAI-KirAV, multiple split-ups of PKS modules on separate genes, and swapping in the domain organization. Interestingly, one PKS enzyme, KirAVI, contains internal AT domains. Based on in silico analysis, a route to pyridone formation involving PKS and NRPS steps was postulated. This hypothesis was experimentally proven by feeding studies with [U-13C3(15)N]beta-alanine and NMR and MS analyses of the isolated pure kirromycin.     

Utilization of alternate substrates by the first three modules of the epothilone synthetase assembly line

The epothilones, a family of macrolactone natural products produced by the myxobacterial species Sorangium cellulosum, are of current clinical interest as antitumor agents. Inspection of the structure of the epothilones suggests a hybrid polyketide/nonribosomal peptide biosynthetic origin, and the recent sequencing of the epothilone biosynthetic gene cluster has validated this proposal. Here we have examined unnatural substrates with the first two enzymes of the biosynthetic pathway, EpoA and EpoB, to investigate the enzymatic construction of alternate heterocyclic structures and the subsequent elongation of these products by the third enzyme of the pathway, EpoC. The epothilone biosynthetic machinery can utilize serine to install an oxazole in place of a thiazole in the epothilone structure and will tolerate functionalized donor groups from the EpoA-ACP domain to produce epothilone fragments modified at the C21 position. These studies with the early enzymes of the epothilone biosynthesis cluster suggest that combinatorial biosynthesis may be a viable means for producing a variety of epothilone analogues that incorporate diversity into the heterocycle starter unit.     

Directed evolution of a gatekeeper domain in nonribosomal peptide synthesis

Modular natural products are biosynthesized by series of enzymes that activate, assemble, and process a nascent chain of building blocks. Adenylation domains are gatekeepers in nonribosomal peptide biosynthesis, providing the entry point for assembly of typical peptide-based natural products. We report the directed evolution of an adenylation domain based on a strategy of using a weak, promiscuous activity as a springboard for reprogramming the biosynthetic assembly line. Randomization of residues invoked in a "specificity-conferring code" and selection for a non-native substrate lead to mutant G2.1, favoring smaller amino acids with a specificity change of 10(5): a 170-fold improvement for L-alanine corresponds to a 10(3)-fold decrease for its original substrate (L-phenylalanine). These results establish directed evolution as a method to change gatekeeper domain specificity and suggest that adaptation of modules in combinatorial biosynthesis is achievable with few mutations during evolution.     

Insights into Molecular Assembly of ACCase Heteromeric Complex in Chlorella variabilis—A Homology Modelling, Docking and Molecular Dynamic Simulation Study

Acetyl-CoA carboxylase (ACCase), a biotin-dependent enzyme that catalyses the first committed step of fatty acid biosynthesis, is considered as a potential target for improving lipid accumulation in oleaginous feedstocks, including microalgae. ACCase is composed of three distinct conserved domains, and understanding the structural details of each catalytic domain assumes great significance to gain insights into the molecular basis of the complex formation and mechanism of biotin transport. In the absence of a crystal structure for any single heteromeric ACCase till date, here we report the first heteromeric association model of ACCase from an oleaginous green microalga, Chlorella variabilis, using a combination of homology modelling, docking and molecular dynamic simulations. The binding site of the docked biotin carboxylase (BC) and carboxyltransferase (CT) were predicted to be contiguous but distinct in biotin carboxyl carrier protein (BCCP) molecule. Simulation studies revealed considerable flexibility for the BC and CT domains in the BCCP-bound forms, thus indicating the adaptive behaviour of BCCP. Further, principal component analysis revealed that in the presence of BCCP, the BC and CT domains exhibited an open-state conformation via the outward clockwise rotation of the binding helices. These conformational changes might be responsible for binding of BCCP domain and its translocation to the respective active sites. Various rearrangements of inter-domain hydrogen bonds (H-bonds) contributed to conformational changes in the structures. H-bond interactions between the interacting residue pairs involving Glu201BCCP/Arg255BC and Asp224BCCP/Gln228CT were found to be essential for the intermolecular assembly. The present findings are consistent with previous biochemical studies.     

Characterization of SyrC, an aminoacyltransferase shuttling threonyl and chlorothreonyl residues in the syringomycin biosynthetic assembly line

Syringomycin, a lipopeptidolactone assembled from nine amino acid monomers by four enzymes, SyrB1, SyrB2, SyrC, and SyrE, is a cyclic nonribosomal peptide made by plant-associated Pseudomonas spp. This assembly is unusual because the terminal residue, 4-chlorothreonine, has been proposed to be added in trans since the ninth module of the megasynthetase SyrE lacks an adenylation domain required for Thr/Cl-Thr activation. SyrC is now identified as a Thr/Cl-Thr aminoacyltransferase, shuttling the Thr/Cl-Thr moiety between the pantetheinyl arms of the thiolation domain of SyrB1 and the thiolation domain in module nine of SyrE. SyrC uses Cys224 as a catalytic nucleophile to generate a Thr/Cl-Thr-S-enzyme intermediate during transfer. SyrC joins a growing family of such aminoacyl-shuttling enzymes that also use covalent catalysis to move aminoacyl groups from carrier proteins during coumermycin and coronamic acid biosynthesis.     

Conversion of L-proline to pyrrolyl-2-carboxyl-S-PCP during undecylprodigiosin and pyoluteorin biosynthesis

Several medically and agriculturally important natural products contain pyrrole moieties. Precursor labeling studies of some of these natural products have shown that L-proline can serve as the biosynthetic precursor for these moieties, including those found in coumermycin A(1), pyoluteorin, and one of the pyrroles of undecylprodigiosin. This suggests a novel mechanism for pyrrole biosynthesis. The biosynthetic gene clusters for these three natural products each encode proteins homologous to adenylation (A) and peptidyl carrier protein (PCP) domains of nonribosomal peptide synthetases in addition to novel acyl-CoA dehydrogenases. Here we show that the three proteins from the undecylprodigiosin and pyoluteorin biosynthetic pathways are sufficient for the conversion of L-proline to pyrrolyl-2-carboxyl-S-PCP. This establishes a novel mechanism for pyrrole biosynthesis and extends the hypothesis that organisms use A/PCP pairs to partition an amino acid into secondary metabolism.     

Enhanced macrocyclizing activity of the thioesterase from tyrocidine synthetase in presence of nonionic detergent

Macrocyclization carried out by thioesterase domains of multimodular nonribosomal peptide synthetases (NRPSs) is a key step in the biosynthesis of many biologically active peptides. The thioesterase excised from tyrocidine synthetase is a versatile macrocyclization catalyst and a useful tool for chemoenzymatic synthesis of diverse cyclic peptides. However, its utility is limited by its short lifetime of catalytic activity as well as significant flux of the acyl-enzyme intermediate to hydrolysis. The addition of Brij 58, a nonionic detergent, above the critical micelle concentration, has dramatic effects on enzyme activity: catalytic activity is extended to >60 min and the rate of cyclization (but not hydrolysis) increases 6-fold, resulting in a net 150- to 300-fold increase in cyclic product yields. This enhanced activity allowed enzymatic macrocyclization of a solid phase library of tyrocidine decapeptides to identify acceptable substitutions at the Orn9 position which had previously been inaccessible for diversification.     

Enzymology of acyl chain macrocyclization in natural product biosynthesis

Polyketides and nonribosomal peptides constitute a large and diverse set of natural products with biological activity in microbial survival and pathogenesis, as well as broad pharmacological utility as antineoplastics, antibiotics or immunosupressants. These molecules 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, although alternative strategies are known. The enzymology of these macrocyclization catalysts, their structure, mechanism, and catalytic versatility, is the subject of this review. The diversity of macrocyclic structures accessed by enzyme catalyzed cyclization of linear acyl chains as well as their inherent substrate tolerance suggests their potential utility in reprogramming natural product biosynthesis pathways or accessing novel macrocyclic structures.     

Identification of a type III thioesterase reveals the function of an operon crucial for Mtb virulence

Rv0098 is part of an operon, Rv0096-Rv0101, from Mycobacterium tuberculosis (Mtb) that is essential for Mtb's survival in mouse macrophages. This operon also contains an acyl carrier protein and one of the only two nonribosomal peptide synthases in Mtb. Rv0098 is annotated in the genome as a hypothetical protein and was proposed to be an acyl-coenzyme A (CoA) dehydratase. The structure of Rv0098, together with subsequent biochemical analysis, indicated that Rv0098 is a long-chain fatty acyl-CoA thioesterase (FcoT). However, FcoT lacks a general base or a nucleophile that is always found in the catalytic site of type II and type I thioesterases, respectively. The active site of Mtb FcoT reveals the structural basis for its substrate specificity for long-chain acyl-CoA and allows us to propose a catalytic mechanism for the enzyme. The characterization of Mtb FcoT provides a putative function of this operon that is crucial for Mtb pathogenicity.     

Nonribosomal biosynthesis of fusaricidins by Paenibacillus polymyxa PKB1 involves direct activation of a D-amino acid

Paenibacillus polymyxa PKB1 produces fusaricidins, a family of lipopeptide antibiotics that strongly inhibits the growth of many plant pathogenic fungi. The fusaricidin biosynthetic gene cluster was cloned and sequenced, and it spans 32.4 kb, including an open reading frame (fusA) encoding a six-module nonribosomal peptide synthetase. The second, fourth, and fifth modules of fusaricidin synthetase each contain an epimerization domain, consistent with the structure of fusaricidins. However, no epimerization domain is found in the sixth module, corresponding to D-Ala. This sixth adenylation domain was produced at a high level in Escherichia coli and is shown to activate D-Ala specifically, providing evidence for direct activation of a D-amino acid by a prokaryotic peptide synthetase. The fusaricidin gene cluster also includes genes involved in the biosynthesis of the lipid moiety, but no genes for resistance, regulation, or transport functions were encountered.     

Kinetic and regiospecific interrogation of covalent intermediates in the nonribosomal peptide synthesis of yersiniabactin

For interrogation of enzyme-bound intermediates in nonribosomal peptide synthetases (NRPSs), mass spectrometry is used to read out the kinetics and substrate specificity of this medicinally important class of enzymes. The protein HMWP2 (230 kDa) catalyzes 11 chemical reactions, four of which could be resolved by fast quench approaches combined with mass spectrometry. The rate of complex intermediate accumulation at the PCP1 active site was observed to occur with a rate of 19 s(-1), with the rate of cysteine acylation faster than that of intermediate translocation. Use of alternative substrates for salicylic acid (at the ArCP carrier domain) and l-cysteine (at the PCP1 carrier domain) revealed a high penalty for omission of the salicyl alcohol. For some substrates, large discrepancies were found between prior adenylation assays and the current MS-based readouts. Indirect evidence for condensation via a thiolate attack (vs an amino group) was also accumulated. This is the first report to correlate the percent occupancy of multiple active sites in parallel with kinetic and structural resolution of intermediates and provides new evidence of interdomain and intermodule communication within thiotemplate assembly lines.     

Biochemical characterization of peptidyl carrier protein (PCP), the thiolation domain of multifunctional peptide synthetases

Background: A structurally diverse group of bioactive peptides is synthesized by peptide synthetases which act as templates for a growing peptide chain, attached to the enzyme via a thloester bond. The protein templates are composed of distinctive substrate-activating modules, whose order dictates the primary structure of the corresponding peptide product. Each module contains defined domains that catalyze adenylation, thioester and peptide bond formation, as well as substrate modifications. To show that a putative thiolation domain (PCP) is involved in covalent binding and transfer of amino aryl residues during non-ribosomal peptide synthesis, we have cloned and biochemically characterized that region of tyrocidine synthetase 1, TycA.Results: The 327-bp gene fragment encoding PCP was cloned using its homology to the genes for the acyl carrier proteins of fatty acid and polyketide biosynthesis. The protein was expressed as a His₆, fusion protein, and purified in a single step by affinity chromatography. Incorporation of β-[³H]alanine, a precursor of coenzyme A, demonstrated the modification of PCP with the cofactor 4′-phosphopantetheine. When an adenylation domain is present to supply the amino adenylate moiety, PCP can be acylated in vitro.Conclusions: PCP can bind covalently to the cofactor phosphopantetheine and can subsequently be acylated, strongly supporting the multiple carrier model of non-ribosomal peptide synthesis. The adenylation and thiolation domains can each act as independent multifunctional enzymes, further confirming the modular structure of peptide synthees, and can also perform sequential steps in trans, as do multienzyme complexes.