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Substrate Trapping in the Siderophore Tailoring Enzyme PvdQ.

ABSTRACT: Siderophore biosynthesis by Pseudomonas aeruginosa enhances virulence and represents an attractive drug target. PvdQ functions in the type-1 pyoverdine biosynthetic pathway by removing a myristoyl anchor from a pyoverdine precursor, allowing eventual release from the periplasm. A circularly permuted version of PvdQ bypasses the self-processing step of this Ntn-hydrolase and retains the activity, selectivity, and structure of wild-type PvdQ, as revealed by a 1.8 Å resolution X-ray crystal structure. A 2.55 Å resolution structure of the inactive S1A/N269D-cpPvdQ mutant in complex with the pyoverdine precursor PVDIq reveals a specific binding pocket for the d-Tyr of this modified peptide substrate. To our knowledge, this structure is the first of a pyoverdine precursor peptide bound to a biosynthetic enzyme. Details of the observed binding interactions have implications for control of pyoverdine biosynthesis and inform future drug design efforts.

SUBMITTER: Clevenger KD

PROVIDER: S-EPMC5373092 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Substrate Trapping in the Siderophore Tailoring Enzyme PvdQ.

Clevenger Kenneth D KD Mascarenhas Romila R Catlin Daniel D Wu Rui R Kelleher Neil L NL Drake Eric J EJ Gulick Andrew M AM Liu Dali D Fast Walter W

ACS chemical biology 20170215 3

Siderophore biosynthesis by Pseudomonas aeruginosa enhances virulence and represents an attractive drug target. PvdQ functions in the type-1 pyoverdine biosynthetic pathway by removing a myristoyl anchor from a pyoverdine precursor, allowing eventual release from the periplasm. A circularly permuted version of PvdQ bypasses the self-processing step of this Ntn-hydrolase and retains the activity, selectivity, and structure of wild-type PvdQ, as revealed by a 1.8 Å resolution X-ray crystal structu ...[more]

PMID: 28186406

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Project description:Anisomycin (compound 1), a pyrrolidine antibiotic, exhibits diverse biological and pharmacologic activities. The biosynthetic gene cluster of compound 1 has been identified previously, and the multistep assembly of the core benzylpyrrolidine scaffold was characterized. However, enzymatic modifications, such as acylation, involved in compound 1 biosynthesis are unknown. In this study, the genetic manipulation of aniI proved that it encoded an indispensable acetyltransferase for compound 1 biosynthesis. Bioinformatics analysis suggested AniI as a member of maltose (MAT) and galactoside O-acetyltransferases (GAT) with C-terminal left-handed parallel beta-helix (LbH) subdomain, which were referred to as LbH-MAT-GAT sugar O-acetyltransferases. However, the biochemical assay identified that its target site was the hydroxyl group of the pyrrolidine ring. AniI was found to be tolerant of acyl donors with different chain lengths for the biosynthesis of compound 1 and derivatives 12 and 13 with butyryl and isovaleryl groups, respectively. Meanwhile, it showed comparable activity toward biosynthetic intermediates and synthesized analogues, suggesting promiscuity to the pyrrolidine ring structure of compound 1. These data may inspire new viable synthetic routes for the construction of more complex pyrrolidine ring scaffolds in compound 1. Finally, the overexpression of aniI under the control of strong promoters contributed to the higher productivities of compound 1 and its analogues. These findings reported here not only improve the understanding of anisomycin biosynthesis but also expand the substrate scope of O-acetyltransferase working on the pyrrolidine ring and pave the way for future metabolic engineering construction of high-yield strains. IMPORTANCE Acylation is an important tailoring reaction during natural product biosynthesis. Acylation could increase the structural diversity and affect the chemical stability, volatility, biological activity, and even the cellular localization of specialized compounds. Many acetyltransferases have been reported in natural product biosynthesis. The typical example of the LbH-MAT-GAT sugar O-acetyltransferase subfamily was reported to catalyze the coenzyme A (CoA)-dependent acetylation of the 6-hydroxyl group of sugars. However, no protein of this family has been characterized to acetylate a nonsugar secondary metabolic product. Here, AniI was found to catalyze the acylation of the hydroxyl group of the pyrrolidine ring and be tolerant of diverse acyl donors and acceptors, which made the biosynthesis more efficient and exclusive for biosynthesis of compound 1 and its derivatives. Moreover, the overexpression of aniI serves as a successful example of genetic manipulation of a modification gene for the high production of final products and might set the stage for future metabolic engineering.

Dataset Information

Substrate Trapping in the Siderophore Tailoring Enzyme PvdQ.

Publications

Substrate Trapping in the Siderophore Tailoring Enzyme PvdQ.

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