Studies on Pseudomonic Acid B Formation in the Biosynthesis of Mupirocin

Abstract

The biosynthesis of the potent antibiotic mupirocin, which consists of a mixture of pseudomonic acids (PAs) isolated from Pseudomonas fluorescens NCIMB 10586, has been studied for decades. While intensive research has unpicked many intricacies of this biosynthetic pathway, the timing and mechanism of several final steps remain elusive. In particular, these include the biosynthetic transformations that follow assembly of the linear pre-monic acid backbone and generate the first genuine mupirocin component, PA-B. The aim therefore was to synthesise an authentic pre-monic acid substrate and combine this with a key 13C-label. Together, this would enable several mechanistic steps to be studied in vitro using both MS and NMR.
The synthesis of a 13C-labelled mimic of pre-monic acid was successfully achieved. The synthetic route generated two further substrates which were accessed en route to this target.
A series of bioassays involving substrates loaded onto ACPs revealed how pre-monic acid is transferred between two multi-enzyme modules, MmpA and MmpE. Once transferred to MmpE, the pre-monic acid mimic was successfully epoxidised in vitro by an isolated oxidoreductase domain on MmpE. Further assays with a putative reverse esterase, MupB, failed to demonstrate esterification with 9HN. before epoxidation with an oxidoreductase domain on MmpE. The second bioassays investigated the unassigned enzyme, MupB, which is proposed to esterify pre-monic acid with 9HN. These may suggest that pre-monic acid undergoes THP ring formation prior to esterification.

Date of Award	1 Oct 2024
Original language	English
Awarding Institution	University of Bristol
Supervisor	Chris L Willis (Supervisor) & Matthew P Crump (Supervisor)