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Specificity of Nonribosomal Peptide Synthetases in the Biosynthesis of the Pseudomonas virulence factor.

ABSTRACT: The Pseudomonas virulence factor (pvf) biosynthetic operon has been implicated in bacterial virulence and signaling. We identified 308 bacterial strains containing pvf homologues that likely produce signaling molecules with distinct structures and biological activities. Several homologues of the nonribosomal peptide synthetase (NRPS), PvfC, were biochemically characterized and shown to activate l-Val or l-Leu. The amino acid selectivity of PvfC and its homologues likely direct pvf signaling activity. We explored the natural diversity of the active site residues present in 92% of the adenylation domains of PvfC homologues and identified key residues for substrate selection and catalysis. Sequence similarity network (SSN) analysis revealed grouping of PvfC homologues that harbor the same active site residues and activate the same amino acids. Our work identified PvfC as a gatekeeper for the structure and bioactivity of the pvf-produced signaling molecules. The combination of active site residue identification and SSN analysis can improve the prediction of aliphatic amino acid substrates for NRPS adenylation domains.

SUBMITTER: Morgan GL

PROVIDER: S-EPMC7179087 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Specificity of Nonribosomal Peptide Synthetases in the Biosynthesis of the Pseudomonas virulence factor.

Morgan Gina L GL Kretsch Ashley M AM Santa Maria Kevin C KC Weeks Savannah J SJ Li Bo B

Biochemistry 20190617 52

The Pseudomonas virulence factor (pvf) biosynthetic operon has been implicated in bacterial virulence and signaling. We identified 308 bacterial strains containing pvf homologues that likely produce signaling molecules with distinct structures and biological activities. Several homologues of the nonribosomal peptide synthetase (NRPS), PvfC, were biochemically characterized and shown to activate l-Val or l-Leu. The amino acid selectivity of PvfC and its homologues likely dire ...[more]

PMID: 31243997

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Project description:BackgroundMost filamentous ascomycete fungi produce high affinity iron chelators called siderophores, biosynthesized nonribosomally by multimodular adenylating enzymes called nonribosomal peptide synthetases (NRPSs). While genes encoding the majority of NRPSs are intermittently distributed across the fungal kingdom, those encoding ferrichrome synthetase NRPSs, responsible for biosynthesis of ferrichrome siderophores, are conserved, which offers an opportunity to trace their evolution and the genesis of their multimodular domain architecture. Furthermore, since the chemistry of many ferrichromes is known, the biochemical and structural 'rules' guiding NRPS substrate choice can be addressed using protein structural modeling and evolutionary approaches.ResultsA search of forty-nine complete fungal genome sequences revealed that, with the exception of Schizosaccharomyces pombe, none of the yeast, chytrid, or zygomycete genomes contained a candidate ferrichrome synthetase. In contrast, all filamentous ascomycetes queried contained at least one, while presence and numbers in basidiomycetes varied. Genes encoding ferrichrome synthetases were monophyletic when analyzed with other NRPSs. Phylogenetic analyses provided support for an ancestral duplication event resulting in two main lineages. They also supported the proposed hypothesis that ferrichrome synthetases derive from an ancestral hexamodular gene, likely created by tandem duplication of complete NRPS modules. Recurrent losses of individual domains or complete modules from this ancestral gene best explain the diversity of extant domain architectures observed. Key residues and regions in the adenylation domain pocket involved in substrate choice and for binding the amino and carboxy termini of the substrate were identified.ConclusionIron-chelating ferrichrome synthetases appear restricted to fission yeast, filamentous ascomycetes, and basidiomycetes and fall into two main lineages. Phylogenetic analyses suggest that loss of domains or modules led to evolution of iterative biosynthetic mechanisms that allow flexibility in biosynthesis of the ferrichrome product. The 10 amino acid NRPS code, proposed earlier, failed when we tried to infer substrate preference. Instead, our analyses point to several regions of the binding pocket important in substrate choice and suggest that two positions of the code are involved in substrate anchoring, not substrate choice.

Dataset Information

Specificity of Nonribosomal Peptide Synthetases in the Biosynthesis of the Pseudomonas virulence factor.

Publications

Specificity of Nonribosomal Peptide Synthetases in the Biosynthesis of the <i>Pseudomonas</i> <i>virulence factor</i>.

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