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.

Giant polyketide synthase enzymes biosynthesize a giant marine polyether biotoxin

“…S20). While five of the “β”-hydroxyls originate from malonate extender units, the out-of-sequence “α”-hydroxy group at C77 is instead likely installed by the α-hydroxylase flavoprotein (FLX) domain ( 27, 28 ) contained in M6. PKZILLA-1 harbors two additional FLX-domain containing modules, M9 and M11, both of which align with the installation of the further α-hydroxylations at C71 and C67, respectively.…”