N-acylation during glidobactin biosynthesis by the tridomain nonribosomal peptide synthetase module GlbF

Glidobactins are hybrid NRPS-PKS natural products that function as irreversible proteasome inhibitors. A?variety of medium chain 2(E),4(E)-diene fatty acids N-acylate the peptidolactam core and contribute significantly to the potency of proteasome inhibition. We have expressed the initiation NRPS module GlbF (C-A-T) in Escherichia coli and observe soluble active protein only on coexpression with the 8?kDa MbtH-like protein, GlbE. Following adenylation and installation of Thr as a T-domain thioester, the starter condensation domain utilizes fatty acyl-CoA donors to acylate the Thr(1) amino group and generate the fatty acyl-Thr(1)-S-pantetheinyl-GlbF intermediate to be used in subsequent chain elongation. Previously proposed to be mediated via acyl carrier protein fatty acid donors, direct utilization of fatty acyl-CoA donors for N-acylation of T-domain tethered amino acids is likely a common strategy for chain initiation in NRPS-mediated lipopeptide biosynthesis.     

Fungal siderophore biosynthesis catalysed by an iterative nonribosomal peptide synthetase

Siderophores play a vital role in the viability of fungi and are essential for the virulence of many pathogenic fungal species. Despite their importance in fungal physiology and pathogenesis, the programming rule of siderophore assembly by fungal nonribosomal peptide synthetases (NRPSs) remains unresolved. Here, we report the characterization of the bimodular fungal NRPS, SidD, responsible for construction of the extracellular siderophore fusarinine C. The use of intact protein mass spectrometry, together with in vitro biochemical assays of native and dissected enzymes, provided snapshots of individual biosynthetic steps during NPRS catalysis. The adenylation and condensation domain of SidD can iteratively load and condense the amino acid building block cis-AMHO, respectively, to synthesize fusarinine C. Our study showcases the iterative programming features of fungal siderophore-producing NRPSs.

Snapshots of fungal siderophore biosynthesis on the biosynthetic assembly-line captured by intact protein mass-spectrometry.     

A new polyketide synthase nonribosomal peptide synthetase hybrid metabolite from plant endophytic fungus Periconia sp.

A new polyketide synthase–nonribosomal peptide synthetase(PKS–NRPS) hybrid metabolite, named pericoannosin B(1), was isolated from endophytic fungus Periconia sp. F-31 of the medicinal plant Annona muricata. The structure and absolute configuration were elucidated by means of extensive spectroscopic analyses(HRMS, NMR, IR, and CD).     

Priming type II polyketide synthases via a type II nonribosomal peptide synthetase mechanism

Benzoic acid priming of the enterocin and actinorhodin type II polyketide synthase complexes was accomplished in vitro via an unprecedented type II nonribosomal peptide synthetase-like mechanism involving the benzoate:acyl carrier protein (ACP) ligase EncN and the ACP EncC. The transfer of the aryl acid to the ACP is ATP-dependent, yet coenzyme A-independent, as characterized with radiolabeled substrates and protein mass spectrometry. Subsequent transport of the ACP-bound aryl group to the native enterocin and the aberrant actinorhodin ketosynthase chain length factor heterodimers was further demonstrated, thereby demonstrating the potential of this biocatalyst for engineering diverse aryl-primed aromatic polyketide agents.     

Directed evolution of the nonribosomal peptide synthetase AdmK generates new andrimid derivatives in vivo

Many lead compounds in the search for new drugs derive from peptides and polyketides whose similar biosynthetic enzymes have been difficult to engineer for production of new derivatives. Problems with generating multiple analogs in a single experiment along with lack of high-throughput methods for structure-based screening have slowed progress in this area. Here, we use directed evolution and a multiplexed assay to screen a library of >14,000 members to generate three derivatives of the antibacterial compound, andrimid. Another limiting factor in reengineering these mega-enzymes of secondary metabolism has been that commonly used hosts such as Escherichia coli often give lower product titers, so our reengineering was performed in the native producer, Pantoea agglomerans. This integrated in?vivo approach can be extended to larger enzymes to create analogs of natural products for bioactivity testing.     

Predictive, structure-based model of amino acid recognition by nonribosomal peptide synthetase adenylation domains

BACKGROUND: Nonribosomal peptide synthetases (NRPSs) are large modular proteins that selectively bind, activate and condense amino acids in an ordered manner. Substrate recognition and activation occurs by reaction with ATP within the adenylation (A) domain of each module. Recently, the crystal structure of the A domain from the gramicidin synthetase (GrsA) with L-phenylalanine and adenosine monophosphate bound has been determined. RESULTS: Critical residues in all known NRPS A domains have been identified that align with eight binding-pocket residues in the GrsA A domain and define sets of remarkably conserved recognition templates. Phylogenetic relationships among these sets and the likely specificity determinants for polar and nonpolar amino acids were determined in light of extensive published biochemical data for these enzymes. The binding specificity of greater than 80% of the known NRPS A domains has been correlated with more than 30 amino acid substrates. CONCLUSIONS: The analysis presented allows the specificity of A domains of unknown function (e.g. from polymerase chain reaction amplification or genome sequencing) to be predicted. Furthermore, it provides a rational framework for altering of A domain specificity by site-directed mutagenesis, which has significant potential for engineering the biosynthesis of novel natural products.     

Preparation of new halogenated diphenyl pyrazine analogs in Escherichia coli by a mono-module fungal nonribosomal peptide synthetase from Penicillium herquei

Pyrazines are important structures widely found in many known drugs. The biological approaches for their synthesis were poorly applied. Herein, microbial production of several halogenated diphenyl pyrazines is reported. These compounds are accumulated via feeding corresponding precursor analogs to Escherichia coli expressing a fungal non-ribosomal peptide synthetase HqlA. Substrate specificity of HqlA was also determined by comparing substrate incorporation efficiencies. HqlA requires a C4-hydroxyl in the substrate and can tolerate certain degrees of size change on the substitution at the carbon next to the hydroxyl group.     

Structural basis for phosphopantetheinyl carrier domain interactions in the terminal module of nonribosomal peptide synthetases

Phosphopantetheine-modified carrier domains play a central role in the template-directed, biosynthesis of several classes of primary and secondary metabolites. Fatty acids, polyketides, and nonribosomal peptides are constructed on multidomain enzyme assemblies using phosphopantetheinyl thioester-linked carrier domains to traffic and activate building blocks. The carrier domain is a dynamic component of the process, shuttling pathway intermediates to sequential enzyme active sites. Here, we report an approach to structurally fix carrier domain/enzyme constructs suitable for X-ray crystallographic analysis. The structure of a two-domain construct of Escherichia coli EntF was determined with a conjugated phosphopantetheinyl-based inhibitor. The didomain structure is locked in an active orientation relevant to the chemistry of nonribosomal peptide biosynthesis. This structure provides details into the interaction of phosphopantetheine arm with the carrier domain and the active site of the thioesterase domain.     

Leinamycin biosynthesis revealing unprecedented architectural complexity for a hybrid polyketide synthase and nonribosomal peptide synthetase

A 135,638 bp DNA region that encompasses the leinamycin (LNM) biosynthetic gene cluster was sequenced from Streptomyces atroolivaceus S-140. The boundaries of the lnm cluster were defined by systematic inactivation of open reading frames within the sequenced region. The lnm cluster spans 61.3 kb of DNA and consists of 27 genes encoding nonribosomal peptide synthetase (NRPS), polyketide synthase (PKS), hybrid NRPS-PKS, resistance, regulatory, and tailoring enzymes, as well as proteins of unknown function. A model for LNM biosynthesis is proposed, central to which is the LNM hybrid NRPS-PKS megasynthetase consisting of discrete (LnmQ and LnmP) and modular (LnmI) NRPS, acyltransferase-less PKS (LnmG, LnmI, and LnmJ), and PKS modules with unusual domain organization. These studies unveil an unprecedented architectural complexity for the LNM hybrid NRPS-PKS megasynthetase and set the stage to investigate the molecular basis for LNM biosynthesis.     

A Switch for the transfer of substrate between nonribosomal peptide and polyketide modules of the rifamycin synthetase assembly line

A nonribosomal peptide synthetase (NRPS) loading module and a polyketide synthase (PKS) elongation module catalyze the preliminary steps in the biosynthesis of the rifamycin antibiotics. A benzoate molecule is covalently attached to the phosphopantetheine arm of the thiolation domain of the loading module when its reaction partner methylmalonyl-CoA is absent. Occupancy of the thiolation domain of the elongation module by a methylmalonyl moiety appears to trigger intermodular transfer of benzoate to the ketosynthase domain of the elongation module. This transthiolation event is fast relative to the initial loading of benzoate onto the loading module. It will be of interest to determine if these results are generally true for intermodular acyl transfer in other NRPS-PKS and PKS assembly lines.     

Decreasing the ring size of a cyclic nonribosomal peptide antibiotic by in-frame module deletion in the biosynthetic genes

Many natural products of therapeutical and biotechnological importance are nonribosomally synthesized peptides. Structural hallmarks of this class of compounds are the occurrence of unusual amino acids, mostly cyclic peptide backbones, and numerous further modifications such as acylation, heterocyclic ring formation, and glycosylation. Because of their structural complexity, chemical synthesis is usually an unattractive route to these molecules. In contrast, genetic engineering of the biosynthesis genes emerges as a potentially powerful approach to the combinatorial biosynthesis of useful analogues of the lead compounds. Nonribosomal peptide synthetases (NRPSs) carry out a sequential multistep assembly and modification of the peptides in a thiotemplate process described by the multiple carrier model. The modular architecture of NRPSs suggests straightforward methods for the reprogramming of these enzymes by exchange of catalytic subunits. However, many of the reported engineering attempts faced low product yields or even inactive hybrid enzymes. Using a new approach to obtain hybrid NRPSs, we show here that the deletion of an entire module in an NRPS assembly line caused the secretion of the predicted peptide antibiotic variant with a decreased ring size. Furthermore, a module exchange resulted in a significantly higher product yield than that observed in previous studies.     

Yersiniabactin synthetase: a four-protein assembly line producing the nonribosomal peptide/polyketide hybrid siderophore of Yersinia pestis

Yersiniabactin synthetase comprises four proteins, YbtE, HMWP1, HMWP2, and YbtU, encompassing seventeen functional domains, twelve catalytic and five carrier, to select, activate, and incorporate salicylate, three cysteines, and one malonyl moiety into the iron chelator yersiniabactin (Ybt). In the present study, yersiniabactin has been reconstituted in vitro from the 4 protein assembly line by the use of eight biosynthetic precursors. The rate of one turnover, comprising 22 chemical operations performed by the assembly line to release the completed Ybt molecule, was determined at 1.4 min(-1). During the course of Ybt production, the elongating acyl-S-enzyme chain was shown to transfer across a nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS) interprotein interface and then a PKS/NRPS intraprotein interface. This study on the Ybt synthetase assembly line represents the first complete in vitro reconstitution of a nonribosomal peptide/polyketide hybrid system.     

Chemoenzymatic synthesis of cryptophycin anticancer agents by an ester bond-forming non-ribosomal peptide synthetase module

Cryptophycins (Crp) are a group of cyanobacterial depsipeptides with activity against drug-resistant tumors. Although they have been shown to be promising, further efforts are required to return these highly potent compounds to the clinic through a new generation of analogues with improved medicinal properties. Herein, we report a chemosynthetic route relying on the multifunctional enzyme CrpD-M2 that incorporates a 2-hydroxy acid moiety (unit D) into Crp analogues. CrpD-M2 is a unique non-ribosomal peptide synthetase (NRPS) module comprised of condensation-adenylation-ketoreduction-thiolation (C-A-KR-T) domains. We interrogated A-domain 2-keto and 2-hydroxy acid activation and loading, and KR domain activity in the presence of NADPH and NADH. The resulting 2-hydroxy acid was elongated with three synthetic Crp chain elongation intermediate analogues through ester bond formation catalyzed by CrpD-M2 C domain. Finally, the enzyme-bound seco-Crp products were macrolactonized by the Crp thioesterase. Analysis of these sequential steps was enabled through LC-FTICR-MS of enzyme-bound intermediates and products. This novel chemoenzymatic synthesis of Crp involves four sequential catalytic steps leading to the incorporation of a 2-hydroxy acid moiety in the final chain elongation intermediate. The presented work constitutes the first example where a NRPS-embedded KR domain is employed for assembly of a fully elaborated natural product, and serves as a proof-of-principle for chemoenzymatic synthesis of new Crp analogues.     

Structure of the EntB multidomain nonribosomal peptide synthetase and functional analysis of its interaction with the EntE adenylation domain

Nonribosomal peptide synthetases are modular proteins that operate in an assembly line fashion to bind, modify, and link amino acids. In the E. coli enterobactin NRPS system, the EntE adenylation domain catalyzes the transfer of a molecule of 2,3-dihydroxybenzoic acid to the pantetheine cofactor of EntB. We present here the crystal structure of the EntB protein that contains an N-terminal isochorismate lyase domain that functions in the synthesis of 2,3-dihydroxybenzoate and a C-terminal carrier protein domain. Functional analysis showed that the EntB-EntE interaction was surprisingly tolerant of a number of point mutations on the surface of EntB and EntE. Mutational studies on EntE support our previous hypothesis that members of the adenylate-forming family of enzymes adopt two distinct conformations to catalyze the two-step reactions.     

Macrocyclization strategies in polyketide and nonribosomal peptide biosynthesis

Nonribosomal peptides and polyketides have attracted considerable attention in basic and applied research and have given rise to a multitude of therapeutic agents. The biological activity of many of these complex natural products, including for example the peptide antibiotics daptomycin and bacitracin or the polyketide anticancer agents epothilone and geldanamycin, specifically relies on the macrocyclization of linear acyl chains that form the backbone of these highly valuable molecules. The construction of the linear acyl precursors is accomplished by modular protein templates that follow comparable assembly line logic. As an enzymatic key step, macrocyclization is introduced after the consecutive condensation of amino acid or acyl-CoA building blocks by dedicated catalysts, and the mature product is released from the biosynthetic machinery. The diverse chain termination strategies of nonribosomal peptide and polyketide assembly lines, the structures and mechanisms of the versatile macrocyclization catalysts, and chemoenzymatic approaches for the development of new therapeutics are the focus of this review. Further, it is illustrated that macrocyclization is not restricted to secondary metabolites, but represents a commonly found structural motif of other biologically active proteins and peptides.     

Structure and noncanonical chemistry of nonribosomal peptide biosynthetic machinery

Covering up to January 2012Structural biology has provided significant insights into the complex chemistry and macromolecular organization of nonribosomal peptide synthetases. In addition, novel pathways are continually described, expanding the knowledge of known biosynthetic chemistry.     

Expression of a functional non-ribosomal peptide synthetase module in Escherichia coli by coexpression with a phosphopantetheinyl transferase

Background: Non-ribosomal peptide synthetases (NRPSs) found in bacteria abd fungi are multifunctional enzymes that catalyze the synthesis of a variety of biologically important peptides. These enzymes are composed of modular units, each responsible for the activation of an amino acid to an aminoacyl adenylate and for the subsequent formation of an aminoacyl thioester with the sulfhydryl group of a 4′-phosphopantetheine moiety. Attempts to express these modules in Escherichia coli have resulted in recombinant proteins deficient in 4′-phosphopantetheine. The recent identification of a family of phosphopantetheinyl transferases (P-pant transferases) associated with NRPS have led us to investigate whether coexpression of NRPS modules with P-pant transferases in E. coli would lead to the incorporation of 4′-phosphopantetheine.Results: A truncated module of gramicidin S synthetase, PheAT(His₆), was expressed as a His₆ fusion protein in E. coli with and without Gsp, the P-pant transferase associated with gramicidin S synthetase. Although PheAT(His₆) expressed alone in E. coli catalyzed Phe-AMP formation from Phe and ATP, < 1% was converted to the Phe thioester. In contrast, >80% of the PheAT(His₆) that was coexpressed with Gsp could form the Phe thioester in the presence of Phe and ATP.Conclusions: Our finding indicates the presence of an almost equimolar amount of 4′-phosphopantetheine covalently bound to the NRPS module PheAT(His₆), and that the functional expression of NRPS modules in E. coli is possible, provided that they are coexpressed with an appropriate P-pant transferase.     

An engineered lantibiotic synthetase that does not require a leader peptide on its substrate

Ribosomally synthesized and post-translationally modified peptides are a rapidly expanding class of natural products. They are typically biosynthesized by modification of a C-terminal segment of the precursor peptide (the core peptide). The precursor peptide also contains an N-terminal leader peptide that is required to guide the biosynthetic enzymes. For bioengineering purposes, the leader peptide is beneficial because it allows promiscuous activity of the biosynthetic enzymes with respect to modification of the core peptide sequence. However, the leader peptide also presents drawbacks as it needs to be present on the core peptide and then removed in a later step. We show that fusing the leader peptide for the lantibiotic lacticin 481 to its biosynthetic enzyme LctM allows the protein to act on core peptides without a leader peptide. We illustrate the use of this methodology for preparation of improved lacticin 481 analogues containing non-proteinogenic amino acids.