The structure and function of the alpha-hydroxylation module of the mupirocin polyketide synthase Ash Winter , Nisha Khanizeman, Zhongshu Song, Abigail Barker-Mountford, Luoyi Wang, Christopher Williams, Christine L. Willis and Matthew P. Crump University of Bristol, UK The incorporation of a-hydroxyl moieties are observed in many biologically active polyketide natural products, yet the process of incorporation into polyketide intermediates remain relatively unknown. The formation of an a-hydroxylation module, whereby a trans -acting monooxygenase functions at the juncture between two type I PKS modules is thought to be responsible for a-hydroxylation in several polyketides (1). Mupirocin, a mixture of pseudomonic acids (PA) A-C, is a commercially available antibiotic produced by Pseudomonas fluorescens NCIMB 10586 and consists of a polyketide derived monic acid homologue, harbouring an a-hydroxy moiety at C6, esterified to 9-hydroxynonanoic acid (2, 3). The non-linear process of a-hydroxylation incorporation and it’s timing within the pathway remains unsolved. We report in vivo and in vitro studies on the control and timing of a-hydroxylation by the trans- acting monooxygenase MupA. By using a combination of purified single enzyme components, authentic substrates, electrospray mass spectrometry and gene knock-outs, these assays define the basic architecture of this a-hydroxylation module (Figure 1). References 1. Helfrich EJN, Piel J. Biosynthesis of polyketides by trans-AT polyketide synthases. Natural Product Reports. 2016;33(2):231- 316. 2. Walker PD, Rowe MT, Winter AJ, Weir ANM, Akter N, Wang L, et al. A Priming Cassette Generates Hydroxylated Acyl Starter Units in Mupirocin and Thiomarinol Biosynthesis. ACS Chemical Biology. 2020;15(2):494-503. 3. Thomas CM, Hothersall J, Willis CL, Simpson TJ. Resistance to and synthesis of the antibiotic mupirocin. Nat Rev Microbiol. 2010;8(4):281-9.
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