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Lookup NU author(s): Professor Paul RaceORCiD
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© 2020 American Chemical Society. Mupirocin, a commercially available antibiotic produced by Pseudomonas fluorescens NCIMB 10586, and thiomarinol, isolated from the marine bacterium Pseudoalteromonas sp. SANK 73390, both consist of a polyketide-derived monic acid homologue esterified with either 9-hydroxynonanoic acid (mupirocin, 9HN) or 8-hydroxyoctanoic acid (thiomarinol, 8HO). The mechanisms of formation of these deceptively simple 9HN and 8HO fatty acid moieties in mup and tml, respectively, remain unresolved. To define starter unit generation, the purified mupirocin proteins MupQ, MupS, and MacpD and their thiomarinol equivalents (TmlQ, TmlS and TacpD) have been expressed and shown to convert malonyl coenzyme A (CoA) and succinyl CoA to 3-hydroxypropionoyl (3-HP) or 4-hydroxybutyryl (4-HB) fatty acid starter units, respectively, via the MupQ/TmlQ catalyzed generation of an unusual bis-CoA/acyl carrier protein (ACP) thioester, followed by MupS/TmlS catalyzed reduction. Mix and match experiments show MupQ/TmlQ to be highly selective for the correct CoA. MacpD/TacpD were interchangeable but alternate trans-acting ACPs from the mupirocin pathway (MacpA/TacpA) or a heterologous ACP (BatA) were nonfunctional. MupS and TmlS selectivity was more varied, and these reductases differed in their substrate and ACP selectivity. The solution structure of MacpD determined by NMR revealed a C-terminal extension with partial helical character that has been shown to be important for maintaining high titers of mupirocin. We generated a truncated MacpD construct, MacpD_T, which lacks this C-terminal extension but retains an ability to generate 3-HP with MupS and MupQ, suggesting further downstream roles in protein-protein interactions for this region of the ACP.
Author(s): Walker PD, Rowe MT, Winter AJ, Weir ANM, Akter N, Wang L, Race PR, Williams C, Song Z, Simpson TJ, Willis CL, Crump MP
Publication type: Article
Publication status: Published
Journal: ACS Chemical Biology
Year: 2020
Volume: 15
Issue: 2
Pages: 494-503
Print publication date: 21/02/2020
Online publication date: 24/01/2020
Acceptance date: 24/01/2020
ISSN (print): 1554-8929
ISSN (electronic): 1554-8937
Publisher: American Chemical Society
URL: https://doi.org/10.1021/acschembio.9b00969
DOI: 10.1021/acschembio.9b00969
PubMed id: 31977176
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