Programmed Iteration Controls the Assembly of the Nonanoic Acid Side Chain of the Antibiotic Mupirocin

Winter, Ashley; Rowe, Matthew; Weir, Angus N. M.; Akter, Nahida; Mbatha, Sbusisiwe Z.; Walker, Paul D.; Williams, Christopher; Song, Zhiyong; Race, Paul R.; Willis, Christine L.; Crump, Matthew P.

doi:10.1002/anie.202212393

“…Using a combination of in vitro and in vivo approaches, many of the complex, nonlinear processing steps within the biosynthetic pathway have been resolved. These include introduction of the 15-methyl group through a β-branching mechanism, [4] THP formation, [5a] epoxidation of the 10,11-alkene, [5b] generation of the 3-hydroxypropionyl fatty acid starter unit, [6] 9-HN biosynthesis, [7] as well as the multi-step conversion of PAÀ B to PAÀ A involving removal of the 8-hydroxy group. [8] A key outstanding question in mupirocin biosynthesis concerns the mechanism of 6-hydroxylation and its precise timing during PA biosynthesis.…”

Section: Introductionmentioning

confidence: 99%

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

Winter,

Khanizeman,

Barker‐Mountford

et al. 2023

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

Winter,

Khanizeman,

Barker‐Mountford

et al. 2023

Self Cite

View full text Add to dashboard Cite

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 Integrated Module Performs Selective ‘Online’ Epoxidation in the Biosynthesis of the Antibiotic Mupirocin

Winter,

de Courcy‐Ireland,

Phillips

et al. 2024

Angewandte Chemie

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The delineation of the complex biosynthesis of the potent antibiotic mupirocin, which consists of a mixture of pseudomonic acids (PAs) isolated from Pseudomonas fluorescens NCIMB 10586, presents significant challenges and the timing and mechanisms of several key transformations remain elusive. Particularly intriguing are the steps that process the linear backbone from the initial polyketide assembly phase to generate the first cyclic intermediate PA‐B. These include epoxidation as well as incorporation of the tetrahydropyran (THP) ring and fatty acid sidechain required for biological activity. Here, we show that the mini‐module MmpE performs a rare online (ACP‐substrate) epoxidation and is integrated (‘in‐cis’) into the polyketide synthase via a docking domain. A linear polyketide fragment with 6 asymmetric centres was synthesised using a convergent approach and used to demonstrate substrate flux via an atypical KS0 and a previously unannotated ACP (MmpE_ACP). MmpE_ACP‐bound synthetic substrates were critical in demonstrating successful epoxidation in vitro by the purified MmpE oxidoreductase domain. Alongside feeding studies, these results confirm the timing as well as chain length dependence of this selective epoxidation. These mechanistic studies pinpoint the location and nature of the polyketide substrate prior to the key formation of the THP ring and esterification that generate PA‐B.

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Programmed Iteration Controls the Assembly of the Nonanoic Acid Side Chain of the Antibiotic Mupirocin

Cited by 7 publications

References 49 publications

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

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

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

An Integrated Module Performs Selective ‘Online’ Epoxidation in the Biosynthesis of the Antibiotic Mupirocin

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