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

Area of Science:

• Biochemistry
• Molecular Biology
• Natural Product Biosynthesis

Background:

• Mupirocin, a vital antibiotic, is synthesized by a trans-AT Type I polyketide synthase (PKS) in Pseudomonas fluorescens.
• The core structure of pseudomonic acid A (PA-A) requires a 6-hydroxy group, likely introduced via alpha-hydroxylation of an acyl carrier protein (ACP)-bound intermediate.

Purpose of the Study:

• To characterize the alpha-hydroxylation bimodule responsible for introducing the 6-hydroxy group in mupirocin biosynthesis.
• To elucidate the enzymatic mechanisms, substrate specificity, and timing of hydroxylation by the MupA enzyme.

Main Methods:

• An in vitro approach combining purified enzyme components with chemical synthesis, isotopic labeling, mass spectrometry, and NMR.
• In vivo studies were integrated to complement in vitro findings.
• Purified enzyme assays were used to determine substrate specificity of MmpA KS^0.

Main Results:

• The first characterization of the alpha-hydroxylation bimodule in the mupirocin pathway is presented.
• Precise timing of hydroxylation by MupA, its substrate specificity, and acyl carrier protein (ACP) dependency were revealed.
• MmpA KS^0 exhibited relaxed substrate specificity, indicating spatiotemporal control of MupA recruitment.

Conclusions:

• The study provides a detailed understanding of the MupA-mediated alpha-hydroxylation step in pseudomonic acid A (PA-A) biosynthesis.
• Intermodular MupA/ACP interactions suggest a mechanism for integrating MupA into the polyketide synthase assembly line.