Structure of a bound peptide phosphonate reveals the mechanism of nocardicin bifunctional thioesterase epimerase-hydrolase half-reactions

Ketan D Patel,Andrew M Gulick,Craig A Townsend,Felipe B D’Andrea,Nicole M Gaudelli,Andrew R Buller

doi:10.1038/s41467-019-11740-6

Abstract

Nonribosomal peptide synthetases (NRPSs) underlie the biosynthesis of many natural products that have important medicinal utility. Protection of the NRPS peptide products from proteolysis is critical to these pathways and is often achieved by structural modification, principally the introduction of d-amino acid residues into the elongating peptide. These amino acids are generally formed in situ from their l-stereoisomers by epimerization domains or dual-function condensation/epimerization domains. In singular contrast, the thioesterase domain of nocardicin biosynthesis mediates both the effectively complete l- to d-epimerization of its C-terminal amino acid residue (≥100:1) and hydrolytic product release. We report herein high-resolution crystal structures of the nocardicin thioesterase domain in ligand-free form and reacted with a structurally precise fluorophosphonate substrate mimic that identify the complete peptide binding pocket to accommodate both stereoisomers. These structures combined with additional functional studies provide detailed mechanistic insight into this unique dual-function NRPS domain.

Highlights

Nonribosomal peptide synthetases (NRPSs) underlie the biosynthesis of many natural products that have important medicinal utility
To complement and further interpret a body of mechanistic studies[10,15,17,18] to understand the unusual steps in nocardicin A biosynthesis, we present structures of this dual-function thioesterase domain NocTE in the unliganded and peptide-bound states
The overall structure of NocTE is similar to known TE domain structures of NRPS and polyketide synthase (PKS) enzymes (Fig. 2)[20]

Summary

Introduction

Nonribosomal peptide synthetases (NRPSs) underlie the biosynthesis of many natural products that have important medicinal utility. While the canonical biosynthesis carried out by NRPSs has been well-studied, an increasing number of examples exist in which NRPS domains are shown to catalyze unusual activities[4] These activities include amide formation by adenylation domains[5,6], condensation domain-catalyzed amide formation between a propionate and a pteridine ring in an unusual “pepteridine” natural product[7], thioesterase domain-mediated condensation of two α-keto carboxylates in the production of quinone or furanone derivatives[8,9], β-lactone and β-lactam cyclization by thioesterase domains[10,11,12], and a Dieckmann condensation catalyzed by an unusual reductase domain, which lacks the conventional catalytic triad, involved in the release of cyclopiazonate tetramic acid neurotoxin[13]. Β-lactam formation was shown to be catalyzed by the condensation (C) domain of the fifth module, using an unusual

Methods

Results

Conclusion