Structure and Function of the α‐Hydroxylation Bimodule of the Mupirocin Polyketide Synthase

Abstract

Mupirocin is a clinically important antibiotic produced by a trans‐AT Type I polyketide synthase (PKS) in Pseudomonas fluorescens. The major bioactive metabolite, pseudomonic acid A (PA‐A), is assembled on a tetrasubstituted tetrahydropyran (THP) core incorporating a 6‐hydroxyl group proposed to be introduced via α‐hydroxylation of the thioester of the acyl carrier protein (ACP) bound polyketide chain. Herein, we describe an in vitro approach combining purified enzyme components, chemical synthesis, isotopic labelling, mass spectrometry and NMR in conjunction with in vivo studies leading to the first characterisation of the α‐hydroxylation bimodule of the mupirocin biosynthetic pathway. These studies reveal the precise timing of hydroxylation by MupA, substrate specificity and the ACP dependency of the enzyme components that comprise this α‐hydroxylation bimodule. Furthermore, using purified enzyme, it is shown that the MmpA KS0 shows relaxed substrate specificity, suggesting precise spatio‐temporal control of in‐trans MupA recruitment versus inter‐module (MmpD to MmpA) transfer. Finally, detection of multiple intermodular‐modular MupA/ACP interactions suggest these bimodules may integrate MupA into their assembly.

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“…An in vitro approach has been used to characterise the α-hydroxylation bimodule of the mupirocin biosynthetic pathway, responsible for the installation of the 6-hydroxy group of pseudomonic acid A 25 . 25 A combination of purified enzyme components, chemical synthesis and labelling studies revealed the precise timing of hydroxylation by MupA and the requirement of a FMN reductase, a complementary acyl carrier protein and a β-keto substrate.…”