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

• Published in: Angewandte Chemie International Edition 2023-11, Vol.62 (47), p.e202312514-n/a
• Main Authors: Winter, Ashley J., Khanizeman, R. Nisha, Barker‐Mountford, Abigail M. C., Devine, Andrew J., Wang, Luoyi, Song, Zhongshu, Davies, Jonathan A., Race, Paul R., Williams, Christopher, Simpson, Thomas J., Willis, Christine L., Crump, Matthew P.
• Format: Article
• Language: English
• Subjects: Acyl carrier protein; Anti-Bacterial Agents - chemistry; Chemical synthesis; Enzymes; Flavin Monooxygenases; Hydroxylation; In vivo methods and tests; Labeling; Mass spectrometry; Mass spectroscopy; Metabolites; Mupirocin; Natural Products; NMR; Nuclear magnetic resonance; Polyketide synthase; Polyketide Synthases - metabolism; Polyketides; Pseudomonas fluorescens; Pseudomonic Acid; Structure-function relationships; Substrate specificity; Substrates
• ISSN: 1433-7851; 1521-3773
• DOI: 10.1002/anie.202312514

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. Mupirocin is a clinically important polyketide antibiotic bearing a 6‐hydroxy group that is essential for biological activity. NMR spectroscopy, authentic synthetic substrates and mass spectrometry were used to establish the mechanistic steps governing incorporation of this key 6‐hydroxy group by an α‐hydroxylation bimodule that recruits a trans acting monooxygenase, MupA.