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

## TL;DR

This study identifies and characterizes the α-hydroxylation bimodule in the mupirocin biosynthesis pathway, revealing its function and enzyme interactions.

## Contribution

First characterization of the α-hydroxylation bimodule in mupirocin biosynthesis using in vitro and in vivo methods.

## Key findings

- MupA performs α-hydroxylation with specific timing and ACP dependency.
- MmpA KS0 exhibits relaxed substrate specificity, indicating spatiotemporal control.
- Intermodular MupA/ACP interactions suggest integration of MupA into the PKS assembly.

## 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‐hydroxy group proposed to be introduced by 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 spatiotemporal control of in trans MupA recruitment in the context of the PKS. Finally, the detection of multiple intermodular MupA/ACP interactions suggests these bimodules may integrate MupA into their assembly.