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Structure and Function of the α-Hydroxylation Bimodule of the Mupirocin Polyketide Synthase |
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| Authors: | Dr Ashley J Winter Dr R Nisha Khanizeman Dr Abigail M C Barker-Mountford Dr Andrew J Devine Dr Luoyi Wang Dr Zhongshu Song Dr Jonathan A Davies Prof Paul R Race Dr Christopher Williams Prof Thomas J Simpson Prof Christine L Willis Prof Matthew P Crump |
| Institution: | 1. School of Chemistry, University of Bristol, Bristol, BS8 1TS UK;2. School of Chemistry, University of Bristol, Bristol, BS8 1TS UK
Contribution: Formal analysis (supporting), ?Investigation (supporting), Methodology (supporting), Writing - review & editing (supporting);3. School of Chemistry, University of Bristol, Bristol, BS8 1TS UK Contribution: ?Investigation (supporting);4. Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101 China;5. School of Chemistry, University of Bristol, Bristol, BS8 1TS UK Contribution: Formal analysis (supporting), ?Investigation (supporting);6. School of Biochemistry, University of Bristol, Bristol, BS8 1TD UK |
| 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. |
| Keywords: | Mupirocin Natural Products Polyketides Pseudomonic Acid Flavin Monooxygenases |