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

Area of Science:

• Biochemistry
• Molecular Biology
• Organic Chemistry

Background:

• Mupirocin, a potent antibiotic produced by Pseudomonas fluorescens, has a complex biosynthesis involving pseudomonic acids (PAs).
• Key steps in PA biosynthesis, including epoxidation and tetrahydropyran (THP) ring formation, remain mechanistically unclear.
• Understanding these steps is crucial for elucidating the complete mupirocin biosynthetic pathway.

Purpose of the Study:

• To investigate the mechanism and timing of epoxidation in mupirocin biosynthesis.
• To identify the specific enzyme and substrate involved in the initial epoxidation step.
• To clarify the integration of the epoxidation module within the polyketide synthase (PKS) assembly line.

Main Methods:

• In vitro biochemical assays using purified MmpE oxidoreductase domain and MmpE_ACP.
• Synthesis of a linear polyketide fragment with six asymmetric centers.
• Feeding studies with Pseudomonas fluorescens NCIMB 10586.

Main Results:

• Demonstrated that the MmpE mini-module performs an 'in-cis' epoxidation on an acyl carrier protein (ACP)-bound substrate.
• Identified a previously unannotated ACP (MmpE_ACP) involved in substrate channeling.
• Confirmed the timing and chain-length dependence of this selective epoxidation via in vitro and feeding studies.

Conclusions:

• The MmpE module is responsible for the crucial early epoxidation step in mupirocin biosynthesis.
• This epoxidation occurs 'online' on an ACP-bound intermediate, preceding THP ring formation.
• Mechanistic insights into this step provide a clearer understanding of PA-B generation.