Generation of D amino acid residues in assembly of arthrofactin by dual condensation/epimerization domains

The first 6 residues of the biosurfactant lipopeptidolactone arthrofactin have the D configuration, yet none of the 11 modules of the nonribosomal peptide synthetase assembly line have epimerization domains. We show that the two-module ArfA subunit and the first module of the ArfB subunit, which act in tandem to produce the N-acyl-D-Leu1-D-Asp2-D-Thr3-S-protein intermediate, activate the L amino acids and epimerize them as the aminoacyl-S-pantetheinyl T domain intermediates before the next downstream condensation. The condensation (C) domains are shown to have (D)C(L) chirality in peptide bond formation. The upstream aminoacyl/peptidyl moiety is epimerized before condensation only when the condensation domains are simultaneously presented with the L-aminoacyl-S-pantetheinyl acceptor. These (D)C(L) catalysts are dual function condensation/epimerization domains that can be predicted by bioinformatics analysis to be responsible for incorporation of all D residues in arthrofactin and of D residues in syringomycin, syringopeptin, and ramoplanin synthetases.     

The specificity-conferring code of adenylation domains in nonribosomal peptide synthetases.

BACKGROUND: Many pharmacologically important peptides are synthesized nonribosomally by multimodular peptide synthetases (NRPSs). These enzyme templates consist of iterated modules that, in their number and organization, determine the primary structure of the corresponding peptide products. At the core of each module is an adenylation domain that recognizes the cognate substrate and activates it as its aminoacyl adenylate. Recently, the crystal structure of the phenylalanine-activating adenylation domain PheA was solved with phenylalanine and AMP, illustrating the structural basis for substrate recognition. RESULTS: By comparing the residues that line the phenylalanine-binding pocket in PheA with the corresponding moieties in other adenylation domains, general rules for deducing substrate specificity were developed. We tested these in silico 'rules' by mutating specificity-conferring residues within PheA. The substrate specificity of most mutants was altered or relaxed. Generalization of the selectivity determinants also allowed the targeted specificity switch of an aspartate-activating adenylation domain, the crystal structure of which has not yet been solved, by introducing a single mutation. CONCLUSIONS: In silico studies and structure-function mutagenesis have defined general rules for the structural basis of substrate recognition in adenylation domains of NRPSs. These rules can be used to rationally alter the specificity of adenylation domains and to predict from the primary sequence the specificity of biochemically uncharacterized adenylation domains. Such efforts could enhance the structural diversity of peptide antibiotics such as penicillins, cyclosporins and vancomycins by allowing synthesis of 'unnatural' natural products.     

Dipeptide formation on engineered hybrid peptide synthetases

BACKGROUND: Nonribosomal peptide synthetases (NRPSs) are modular 'megaenzymes' that catalyze the assembly of a large number of bioactive peptides using the multiple carrier thiotemplate mechanism. The modules comprise specific domains that act as distinct units to catalyze specific reactions associated with substrate activation, modification and condensation. Such an arrangement of biosynthetic templates has evoked interest in engineering novel NRPSs. RESULTS: We describe the design and construction of a set of dimodular hybrid NRPSs. By introducing domain fusions between adenylation and thiolation (PCP) domains we designed synthetic templates for dipeptide formation. The predicted dipeptides, as defined by the specificity and arrangement of the adenylation domains of the constructed templates, were synthesized in vitro. The effect of the intramolecular fusion was investigated by determining kinetic parameters for substrate adenylation and thiolation. The rate of dipeptide formation on the artificial NRPSs is similar to that of natural templates. CONCLUSIONS: Several new aspects concerning the tolerance of NRPSs to domain swaps can be deduced. By choosing the fusion site in the border region of adenylation and PCP domains we showed that the PCP domain exhibits no general substrate selectivity. There was no suggestion that selectivity of the condensation reaction was biased towards the donor amino acid, whereas at the acceptor position there was a size-determined selection. In addition, we demonstrated that a native elongation module can be converted to an initiation module for peptide-bond formation. These results represent the first example of rational de novo synthesis of small peptides on engineered NRPSs.     

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.     

Reprogramming Nonribosomal Peptide Synthetases for “Clickable” Amino Acids

Nonribosomal peptide synthetases (NRPSs) are multifunctional enzymes that produce a wide array of bioactive peptides. Here we show that a single tryptophan‐to‐serine mutation in phenylalanine‐specific NRPS adenylation domains enables the efficient activation of non‐natural aromatic amino acids functionalized with azide and alkyne groups. The resulting 10⁵‐fold switch in substrate specificity was achieved without appreciable loss of catalytic efficiency. Moreover, the effective communication of the modified A domains with downstream modules in dipeptide synthetases permitted incorporation of O‐propargyl‐L ‐tyrosine into diketopiperazines both in vitro and in vivo, even in the presence of competing phenylalanine. Because azides and alkynes readily undergo bioorthogonal click reactions, reprogramming NRPSs to accept non‐natural amino acids that contain these groups provides a potentially powerful means of isolating, labeling, and modifying biologically active peptides.     

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.     

The bacitracin biosynthesis operon of Bacillus licheniformis ATCC 10716: molecular characterization of three multi-modular peptide synthetases

Background: The branched cyclic dodecylpeptide antibiotic bacitracin, produced by special strains of Bacillus, is synthesized nonribosomally by a large multienzyme complex composed of the three bacitracin synthetases BA1, BA2 and BA3. These enzymes activate and incorporate the constituent amino acids of bacitracin by a thiotemplate mechanism in a pathway driven by a protein template. The biochemical features of these enzymes have been studied intensively but little is known about the molecular organization of their genes.Results: The entire bacitracin synthetase operon containing the genes bacA-bacC was cloned and sequenced, identifying a modular structure typical of peptide synthetases. The bacA gene product (BA1, 598 kDa) contains five modules, with an internal epimerization domain attached to the fourth; bacB encodes BA2 (297 kDa), and has two modules and a carboxy-terminal epimerization domain; bacC encodes BA3, five modules (723 kDa) with additional internal epimerization domains attached to the second and fourth. A carboxy-terminal putative thioesterase domain was also detected in BA3. A putative cyclization domain was found in BA1 that may be involved in thiazoline ring formation. The adenylation/thioester-binding domains of the first two BA1 modules were overproduced and the detected amino-acid specificity coincides with the first two amino acids in bacitracin. Disruption of chromosomal bacB resulted in a bacitracin-deficient mutant.Conclusions: The genes encoding the bacitracin synthetases BA1, BA2 and BA3 are organized in an operon, the structure of which reflects the modular architecture expected of peptide synthetases. In addition, a putative thiazoline ring formation domain was identified in the BA1 gene.     

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.     

Exploring modular reengineering strategies to redesign the teicoplanin non-ribosomal peptide synthetase

Non-ribosomal peptide synthesis is an important biosynthesis pathway in secondary metabolism. In this study we have investigated modularisation and redesign strategies for the glycopeptide antibiotic teicoplanin. Using the relocation or exchange of domains within the NRPS modules, we have identified how to initiate peptide biosynthesis and explored the requirements for the functional reengineering of both the condensation/adenylation domain and epimerisation/condensation domain interfaces. We have also demonstrated strategies that ensure communication between isolated NRPS modules, leading to new peptide assembly pathways. This provides important insights into NRPS reengineering of glycopeptide antibiotic biosynthesis and has broad implications for the redesign of other NRPS systems.

Redesign of the non-ribosomal peptide synthetase (NRPS) from teicoplanin biosynthesis has been extensively investigated via domain exchange, interface reengineering and through engineering communication between isolated NRPS modules.     

Characterization of the cereulide NRPS alpha-hydroxy acid specifying modules: activation of alpha-keto acids and chiral reduction on the assembly line

Several nonribosomal peptide natural products are composites of alpha-hydroxy acid and alpha-amino acid monomers. Cereulide, the emetic toxin from the human pathogen Bacillus cereus, and valinomycin, from Streptomyces spp., are closely related macrocyclic K+ ionophores. The macrocyclic core of each natural product contains alternating peptide (six) and ester (six) bonds, and their cyclododecadepsipeptide structures consist of a tetradepsipeptide unit repeated three times. Here we overexpress the cereulide NRPS alpha-hydroxy acid specifying modules from CesA and CesB and demonstrate that each contains an alpha-keto acid activating adenylation domain and a chiral alpha-ketoacyl-S-carrier protein reductase (alpha-KR). The logic used by the cereulide NRPS is likely at work in the valinomycin NRPS and may be the general strategy used in bacterial NRPSs to form alpha-hydroxy acid containing natural products.     

Interdomain communication between the thiolation and thioesterase domains of EntF explored by combinatorial mutagenesis and selection

Thiolation (T) domains are protein way stations in natural product assembly lines. In the enterobactin synthetase, the T domain on EntF is recognized in cis by its catalytic partners: the EntF condensation (C), adenylation (A), and thioesterase (TE) domains. To assess surface features of the EntF T domain recognized by C, A, and TE, regions of the EntF T domain were submitted to shotgun alanine scanning and Ent production selection, which revealed residues that could not be substituted by Ala. EntF mutants bearing Ala in such positions were assayed in vitro for Ent production with EntEB, and for A-T, C-T, and T-TE communications. We concluded that G1027A and M1030A are specifically defective in acyl transfer from T to TE. These residues define an interaction surface between these two in cis domains in an NRPS module.     

Identification and characterization of the lysobactin biosynthetic gene cluster reveals mechanistic insights into an unusual termination module architecture

Lysobactin (katanosin B) is a macrocyclic depsipeptide, displaying high antibacterial activity against human pathogens. In this work, we have identified and characterized the entire biosynthetic gene cluster responsible for lysobactin assembly. Sequential analysis of the Lysobacter sp. ATCC 53042 genome revealed the lysobactin gene cluster to encode two multimodular nonribosomal peptide synthetases. As the number of modules found within the synthetases LybA and LybB directly correlates with the primary sequence of lysobactin, a linear logic of lysobactin biosynthesis is proposed. Investigation of adenylation domain specificities in?vitro confirmed the direct association between the synthetases and lysobactin biosynthesis. Furthermore, an unusual tandem thioesterase architecture of the LybB termination module was identified. Biochemical characterization of the individual thioesterases in?vitro provides evidence that solely penultimate thioesterase domain mediates the cyclization and simultaneous release of lysobactin.     

Protein assembly line components in prodigiosin biosynthesis: characterization of PigA,G,H,I,J

The red streptomycete metabolite prodigiosin has a unique tripyrrolic structure with two of the three pyrrolyl moieties in tandem. Five enzymes, PigA,G,H,I, and J, are involved in dipyrrole (rings A and B) formation. We have heterologously expressed and purified from Escherichia coli these five enzymes. At first, pyrrole ring A is formed on the peptidyl carrier protein PigG by one of two possible ways: (i) by action of the adenylation domain PigI that transforms l-proline into l-prolyl-AMP and by the flavoprotein dehydrogenase PigA responsible for the four-electron oxidation reaction; (ii) by loading with the pyrrolyl-2-carboxyl-(S)-pantetheinyl moiety from synthetic pyrrolyl-CoA using the phosphopantetheinyl transferase Sfp. Subsequently, pyrrole ring B is constructed by PigH after the transfer of ring A to the ketosynthase of PigJ. PigH consists of three domains: two acyl carrier proteins (ACPs) and a seryltransferase (SerT). Using HPLC and nanospray-Fourier Transform Mass Spectrometry (nFTMS), we established that all three domains of PigH undergo post-translational modifications and gained insight into the machinery involved in 2,2-dipyrrole biosynthesis.     

A Synthetic Adenylation‐Domain‐Based tRNA‐Aminoacylation Catalyst

The incorporation of non‐proteinogenic amino acids represents a major challenge for the creation of functionalized proteins. The ribosomal pathway is limited to the 20–22 proteinogenic amino acids while nonribosomal peptide synthetases (NRPSs) are able to select from hundreds of different monomers. Introduced herein is a fusion‐protein‐based design for synthetic tRNA‐aminoacylation catalysts based on combining NRPS adenylation domains and a small eukaryotic tRNA‐binding domain (Arc1p‐C). Using rational design, guided by structural insights and molecular modeling, the adenylation domain PheA was fused with Arc1p‐C using flexible linkers and achieved tRNA‐aminoacylation with both proteinogenic and non‐proteinogenic amino acids. The resulting aminoacyl‐tRNAs were functionally validated and the catalysts showed broad substrate specificity towards the acceptor tRNA. Our strategy shows how functional tRNA‐aminoacylation catalysts can be created for bridging the ribosomal and nonribosomal worlds. This opens up new avenues for the aminoacylation of tRNAs with functional non‐proteinogenic amino acids.     

Mechanistic analysis of acyl transferase domain exchange in polyketide synthase modules

Many polyketides are synthesized by a class of multifunctional enzymes called type I modular polyketide synthases (PKSs). Several reports have described the power of predictively altering polyketide structure by replacing individual PKS domains with homologues from other PKSs. For example, numerous erythromycin analogues have been generated by replacing individual methylmalonyl-specific acyl transferase (AT) domains of the 6-deoxyerythronolide B synthase (DEBS) with malonyl-, ethylmalonyl-, or methoxymalonyl-specific domains. However, the construction of hybrid PKS modules often attenuates product formation both kinetically and distributively. The molecular basis for this mechanistic imperfection is not understood. We have systematically analyzed the impact of replacing an AT domain of DEBS on acyl-AT formation, acyl-CoA:HS-NAc acyl transferase activity, acyl-CoA:ACP acyl transferase activity (nucleophile charging), acyl-SNAc:ketosynthase acyl transferase activity (electrophile charging), and beta-ketoacyl ACP synthase activity (condensation). As usual, domain junctions were located in interdomain regions flanking the AT domain. Kinetic analysis of hybrid modules containing either malonyl transferase or methylmalonyl transferase domains revealed a 15-20-fold decrease in overall turnover numbers of the hybrid modules as compared to the wild-type module. In contrast, both the activity and the specificity of the heterologous AT domains remained unaffected. Moreover, no defects could be detected in the ability of the heterologous AT domains to catalyze acyl-CoA:ACP acyl transfer. Single turnover studies aimed at directly probing the ketosynthase-catalyzed reaction led to two crucial findings. First, wild-type modules catalyzed chain elongation with comparable efficiency regardless of whether methylmalonyl-ACP or malonyl-ACP were the nucleophilic substrates. Second, chain elongation in all hybrid modules tested was seriously attenuated relative to the wild-type module. Our data suggest that, as currently practiced, the most deleterious impact of AT domain swapping is not on the substrate specificity. Rather, it is due to the impaired ability of the KS and ACP domains in the hybrid module to catalyze chain elongation. Consistent with this proposal, limited proteolysis of wild-type and hybrid modules showed major differences in cleavage patterns, especially in the region between the KR and ACP domains.     

A systematic family-wide investigation reveals that ~30% of mammalian PDZ domains engage in PDZ-PDZ interactions

PDZ domains are independently folded modules that typically mediate protein-protein interactions by binding to the C termini of their target proteins. However, in a few instances, PDZ domains have been reported to dimerize with other PDZ domains. To investigate this noncanonical-binding mode further, we used protein microarrays comprising virtually every mouse PDZ domain to systematically query all possible PDZ-PDZ pairs. We then used fluorescence polarization to retest and quantify interactions and coaffinity purification to test biophysically validated interactions in the context of their full-length proteins. Overall, we discovered 37 PDZ-PDZ interactions involving 46 PDZ domains (~30% of all PDZ domains tested), revealing that dimerization is a more frequently used binding mode than was previously appreciated. This suggests that many PDZ domains evolved to form multiprotein complexes by simultaneously interacting with more than one ligand.     

Formation of functional heterologous complexes using subunits from the picromycin, erythromycin and oleandomycin polyketide synthases

BACKGROUND: Recently developed tools for the genetic manipulation of modular polyketide synthases (PKSs) have advanced the development of combinatorial biosynthesis technologies for drug discovery. Although many of the current techniques involve engineering individual domains or modules of the PKS, few experiments have addressed the ability to combine entire protein subunits from different modular PKSs to create hybrid polyketide pathways. We investigated this possibility by in vivo assembly of heterologous PKS complexes using natural and altered subunits from related macrolide PKSs. RESULTS: The pikAI and pikAII genes encoding subunits 1 and 2 (modules 1-4) of the picromycin PKS (PikPKS) and the eryAIII gene encoding subunit 3 (modules 5-6) of the 6-deoxyerythronolide B synthase (DEBS) were cloned in two compatible Streptomyces expression vectors. A strain of Streptomyces lividans co-transformed with the two vectors produced the hybrid macrolactone 3-hydroxynarbonolide. Co-expression of the same pik genes with the gene for subunit 3 of the oleandomycin PKS (OlePKS) was also successful. A series of hybrid polyketide pathways was then constructed by combining PikPKS subunits 1 and 2 with modified DEBS3 subunits containing engineered domains in modules 5 or 6. We also report the effect of junction location in a set of DEBS-PikPKS fusions. CONCLUSIONS: We show that natural as well as engineered protein subunits from heterologous modular PKSs can be functionally assembled to create hybrid polyketide pathways. This work represents a new strategy that complements earlier domain engineering approaches for combinatorial biosynthesis in which complete modules or PKS protein subunits, in addition to individual enzymatic domains, are used as building blocks for PKS engineering.     

Directed Evolution of Piperazic Acid Incorporation by a Nonribosomal Peptide Synthetase**

Engineering of biosynthetic enzymes is increasingly employed to synthesize structural analogues of antibiotics. Of special interest are nonribosomal peptide synthetases (NRPSs) responsible for the production of important antimicrobial peptides. Here, directed evolution of an adenylation domain of a Pro-specific NRPS module completely switched substrate specificity to the non-standard amino acid piperazic acid (Piz) bearing a labile N−N bond. This success was achieved by UPLC-MS/MS-based screening of small, rationally designed mutant libraries and can presumably be replicated with a larger number of substrates and NRPS modules. The evolved NRPS produces a Piz-derived gramicidin S analogue. Thus, we give new impetus to the too-early dismissed idea that widely accessible low-throughput methods can switch the specificity of NRPSs in a biosynthetically useful fashion.     

Structure-based dissociation of a type I polyketide synthase module

Individual modules of modular polyketide synthases (PKSs) such as 6-deoxyerythronolide B synthase (DEBS) consist of conserved, covalently linked domains separated by unconserved intervening linker sequences. To better understand the protein-protein and enzyme-substrate interactions in modular catalysis, we have exploited recent structural insights to prepare stand-alone domains of selected DEBS modules. When combined in vitro, ketosynthase (KS), acyl transferase (AT), and acyl carrier protein (ACP) domains of DEBS module 3 catalyzed methylmalonyl transfer and diketide substrate elongation. When added to a minimal PKS, ketoreductase domains from DEBS modules 1, 2, and 6 showed specificity for the beta-ketoacylthioester substrate, but not for either the ACP domain carrying the polyketide substrate or the KS domain that synthesized the substrate. With insights into catalytic efficiency and specificity of PKS modules, our results provide guidelines for constructing optimal hybrid PKS systems.     

Proteomics-based discovery of koranimine, a cyclic imine natural product

Nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) are large enzymes responsible for the biosynthesis of medically and ecologically important secondary metabolites. In a previous report, we described a proteomics approach to screen for expressed NRPSs or PKSs from bacteria with or without sequenced genomes. Here we used this proteome mining approach to discover a novel natural product arising from rare adenylation (A) and reductase (Red) domains in its biosynthetic machinery. We also cloned the entire gene cluster and elucidated the biosynthesis of the new compound, which is produced by an unsequenced Bacillus sp. isolated from soil collected in Koran, Louisiana.