Characterization of the Ketosynthase and Acyl Carrier Protein Domains at the LnmI Nonribosomal Peptide Synthetase-Polyketide Synthase Interface for Leinamycin Biosynthesis.
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ABSTRACT:
SUBMITTER: Huang Y
PROVIDER: S-EPMC5013926 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Many microbial secondary metabolites are produced by multienzyme complexes comprising nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). The ketosynthase (KS) domains of polyketide synthase normally catalyze the decarboxylative Claisen condensation of acyl and malonyl blocks to extend the polyketide chain. However, the terminal KS domain in tenuazonic acid synthetase 1 (TAS1) from the fungus Pyricularia oryzae conducts substrate cyclization. Here, we report on the unique features of the KS domain in TAS1. We observed that this domain is monomeric, not dimeric as is typical for KSs. Analysis of a 1.68-Å resolution crystal structure suggests that the substrate cyclization is triggered via proton abstraction from the active methylene moiety in the substrate by a catalytic His-322 residue. Additionally, we show that TAS1 KS promiscuously accepts aminoacyl substrates and that this promiscuity can be increased by a single amino acid substitution in the substrate-binding pocket of the enzyme. These findings provide insight into a KS domain that accepts the amino acid-containing substrate in an NRPS-PKS hybrid enzyme and provide hints to the substrate cyclization mechanism performed by the KS domain in the biosynthesis of the mycotoxin tenuazonic acid.
| S-EPMC7450107 | biostudies-literature
Project description:Every polyketide synthase module has an acyl carrier protein (ACP) and a ketosynthase (KS) domain that collaborate to catalyze chain elongation. The same ACP then engages the KS domain of the next module to facilitate chain transfer. Understanding the mechanism for this orderly progress of the growing polyketide chain represents a fundamental challenge in assembly line enzymology. Using both experimental and computational approaches, the molecular basis for KS-ACP interactions in the 6-deoxyerythronolide B synthase has been decoded. Surprisingly, KS-ACP recognition is controlled at different interfaces during chain elongation versus chain transfer. In fact, chain elongation is controlled at a docking site remote from the catalytic center. Not only do our findings reveal a new principle in the modular control of polyketide antibiotic biosynthesis, they also provide a rationale for the mandatory homodimeric structure of polyketide synthases, in contrast to the monomeric nonribosomal peptide synthetases.
| S-EPMC3009775 | biostudies-literature
Project description:Polyketide synthases (PKSs) catalyze the production of numerous biologically important natural products via repeated decarboxylative condensation reactions. Modular PKSs, such as the 6-deoxyerythronolide B synthase (DEBS), consist of multiple catalytic modules, each containing a unique set of covalently linked catalytic domains. To better understand the engineering opportunities of these assembly lines, the extender unit and acyl carrier protein (ACP) specificity of keto synthase (KS) domains from modules 3 and 6 of DEBS were analyzed. These studies were undertaken with a newly developed didomain [KS][AT] construct, which lacks its own ACP domain and can therefore be interrogated with homologous or heterologous ACP or acyl-ACP substrates. By substituting the natural methylmalonyl extender unit with a malonyl group, a modest role was demonstrated for the KS in recognition of the nucleophilic substrate. The KS domain from module 3 of DEBS was found to exhibit a distinct ACP-recognition profile from the KS domain of module 6. On the basis of the above kinetic insights, a hybrid module was constructed ([KS3][AT3][KR5][ACP5][TE]) which displayed substrate recognition and elongation capabilities consistent with the natural module 3 protein. Unlike module 3, however, which lacks a ketoreductase (KR) domain, the hybrid module was able to catalyze reduction of the beta-ketothioester product of chain elongation. The high expression level and functionality of this hybrid protein demonstrates the usefulness of kinetic analysis for hybrid module design.
| S-EPMC2532788 | biostudies-literature
Project description:Biochemical studies of purified and dissected fungal polyketide synthase and nonribosomal peptide synthetase (PKS-NRPS) hybrid enzymes involved in biosynthesis of pseurotin and aspyridone indicate that one α-methylation step during polyketide synthesis is a prerequisite and a key checkpoint for chain transfer between PKS and NRPS modules. In the absence of the resulting γ-methyl feature, the completed polyketide intermediate is offloaded as an α-pyrone instead of being aminoacylated by the NRPS domain. These examples illustrate that precisely timed tailoring domain activities play critical roles in the overall programming of the iterative PKS (and NRPS) functions.
| S-EPMC4275151 | biostudies-literature
Project description:Truncated iterative polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) megasynthases in which only the C domain is present are widespread in fungi, yet nearly all members have unknown functions. Bioinformatics analysis showed that the C domains of such PKS-C enzymes are noncanonical due to substitution at the second histidine in the active site HHxxxDG motif. Here, we used genome mining strategy to characterize a cryptic PKS-C hybrid from Talaromyces wortmanii and discovered the products are reduced long-chain polyketides amidated with a specific ?-amino acid 5-aminopentanoic acid (5PA). The wortmanamides resemble long-chain N-acyl-amide signaling lipids that target diverse receptors including GPCRs. The noncanonical C domain of this PKS-C hybrid was also demonstrated to be a bona fide condensation domain that specifically selects 5PA and catalyzes amidation to release polyketide chain.
| S-EPMC5817892 | biostudies-literature
Project description:Acyl carrier protein (ACP) domains act as interaction hubs within modular polyketide synthase (PKS) systems, employing specific protein-protein interactions to present acyl substrates to a series of enzyme active sites. Many domains from the multimodular PKS that generates the toxin mycolactone display an unusually high degree of sequence similarity, implying that the few sites which vary may do so for functional reasons. When domain boundaries based on prior studies were used to prepare two isolated ACP segments from this system for studies of their interaction properties, one fragment adopted the expected tertiary structure, but the other failed to fold, despite sharing a sequence identity of 49%. Secondary structure prediction uncovered a previously undetected helical region (H0) that precedes the canonical helix-bundle ACP topology in both cases. This article reports the NMR solution structures of two N-terminally extended mycolactone mACP constructs, mH0ACPa and mH0ACPb, both of which possess an additional α-helix that behaves like a rigid component of the domain. The interactions of these species with a phosphopantetheinyl transferase and a ketoreductase domain are unaffected by the presence of H0, but a shorter construct that lacks the H0 region is shown to be substantially less thermostable than mH0ACPb. Bioinformatics analysis suggests that the extended H0-ACP motif is present in 98% of type I cis-acyltransferase PKS chain-extension modules. The polypeptide linker that connects an H0-ACP motif to the preceding domain must therefore be ~12 residues shorter than previously thought, imposing strict limits on ACP-mediated substrate delivery within and between PKS modules.
| S-EPMC6382882 | biostudies-literature
Project description:BackgroundPyridoxal 5'-phosphate (PLP)-dependent enzymes of fold type I, the most studied structural class of the PLP-dependent enzyme superfamily, are known to exist as stand-alone homodimers or homotetramers. These enzymes have been found also embedded in multimodular and multidomain assembly lines involved in the biosynthesis of polyketides (PKS) and nonribosomal peptides (NRPS). The aim of this work is to provide a proteome-wide view of the distribution and characteristics of type I domains covalently integrated in these assemblies in prokaryotes.ResultsAn ad-hoc Hidden Markov profile was calculated using a sequence alignment derived from a multiple structural superposition of distantly related PLP-enzymes of fold type I. The profile was utilized to scan the sequence databank and to collect the proteins containing at least one type I domain linked to a component of an assembly line in bacterial genomes. The domains adjacent to a carrier protein were further investigated. Phylogenetic analysis suggested the presence of four PLP-dependent families: Aminotran_3, Beta_elim_lyase and Pyridoxal_deC, occurring mainly within mixed NRPS/PKS clusters, and Aminotran_1_2 found mainly in PKS clusters. Sequence similarity to the reference PLP enzymes with solved structures ranged from 24 to 42% identity. Homology models were built for each representative type I domain and molecular docking simulations with putative substrates were carried out. Prediction of the protein-protein interaction sites evidenced that the surface regions of the type I domains embedded within multienzyme assemblies were different from those of the self-standing enzymes; these structural features appear to be required for productive interactions with the adjacent domains in a multidomain context.ConclusionsThis work provides a systematic view of the occurrence of type I domain within NRPS and PKS assembly lines and it predicts their structural characteristics using computational methods. Comparison with the corresponding stand-alone enzymes highlighted the common and different traits related to various aspects of their structure-function relationship. Therefore, the results of this work, on one hand contribute to the understanding of the functional and structural diversity of the PLP-dependent type I enzymes and, on the other, pave the way to further studies aimed at their applications in combinatorial biosynthesis.
| S-EPMC3870968 | biostudies-literature
Project description:Polyketides are a medicinally important class of natural products. The architecture of modular polyketide synthases (PKSs), composed of multiple covalently linked domains grouped into modules, provides an attractive framework for engineering novel polyketide-producing assemblies. However, impaired domain-domain interactions can compromise the efficiency of engineered polyketide biosynthesis. To facilitate the study of these domain-domain interactions, we have used nuclear magnetic resonance (NMR) spectroscopy to determine the first solution structure of an acyl carrier protein (ACP) domain from a modular PKS, 6-deoxyerythronolide B synthase (DEBS). The tertiary fold of this 10-kD domain is a three-helical bundle; an additional short helix in the second loop also contributes to the core helical packing. Superposition of residues 14-94 of the ensemble on the mean structure yields an average atomic RMSD of 0.64 +/- 0.09 Angstrom for the backbone atoms (1.21 +/- 0.13 Angstrom for all non-hydrogen atoms). The three major helices superimpose with a backbone RMSD of 0.48 +/- 0.10 Angstrom (0.99 +/- 0.11 Angstrom for non-hydrogen atoms). Based on this solution structure, homology models were constructed for five other DEBS ACP domains. Comparison of their steric and electrostatic surfaces at the putative interaction interface (centered on helix II) suggests a model for protein-protein recognition of ACP domains, consistent with the previously observed specificity. Site-directed mutagenesis experiments indicate that two of the identified residues influence the specificity of ACP recognition.
| S-EPMC2204127 | biostudies-literature
Project description:To identify the species of butyrolactol-producing Streptomyces strain TP-A0882, whole genome-sequencing of three type strains in a close taxonomic relationship was performed. In silico DNA-DNA hybridization using the genome sequences suggested that Streptomyces sp. TP-A0882 is classified as Streptomyces diastaticus subsp. ardesiacus. Strain TP-A0882, S. diastaticus subsp. ardesiacus NBRC 15402T, Streptomyces coelicoflavus NBRC 15399T, and Streptomyces rubrogriseus NBRC 15455T harbor at least 14, 14, 10, and 12 biosynthetic gene clusters (BGCs), respectively, coding for nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). All 14 gene clusters were shared by S. diastaticus subsp. ardesiacus strains TP-A0882 and NBRC 15402T, while only four gene clusters were shared by the three distinct species. Although BGCs for bacteriocin, ectoine, indole, melanine, siderophores such as deferrioxamine, terpenes such as albaflavenone, hopene, carotenoid and geosmin are shared by the three species, many BGCs for secondary metabolites such as butyrolactone, lantipeptides, oligosaccharide, some terpenes are species-specific. These results indicate the possibility that strains belonging to the same species possess the same set of secondary metabolite-biosynthetic pathways, whereas strains belonging to distinct species have species-specific pathways, in addition to some common pathways, even if the strains are taxonomically close.
| S-EPMC5932044 | biostudies-literature
Project description:(1) Background: Phytohabitans is a recently established genus belonging to rare actinomycetes. It has been unclear if its members have the capacity to synthesize diverse secondary metabolites. Polyketide and nonribosomal peptide compounds are major secondary metabolites in actinomycetes and expected as a potential source for novel pharmaceuticals. (2) Methods: Whole genomes of Phytohabitans flavus NBRC 107702T, Phytohabitans rumicis NBRC 108638T, Phytohabitans houttuyneae NBRC 108639T, and Phytohabitans suffuscus NBRC 105367T were sequenced by PacBio. Polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) gene clusters were bioinformatically analyzed in the genome sequences. (3) Results: These four strains harbored 10, 14, 18 and 14 PKS and NRPS gene clusters, respectively. Most of the gene clusters were annotated to synthesis unknown chemistries. (4) Conclusions: Members of the genus Phytohabitans are a possible source for novel and diverse polyketides and nonribosomal peptides.
| S-EPMC7692728 | biostudies-literature