Characterization of the Flavin-Dependent Monooxygenase Involved in the Biosynthesis of the Nocardiosis-Associated Polyketide†

Some species of the Nocardia genus harbor a highly conserved biosynthetic gene cluster designated as the NOCardiosis-Associated Polyketide (NOCAP) synthase that produces a unique glycolipid natural product. The NOCAP glycolipid is composed of a fully substituted benzaldehyde headgroup linked to a polyfunctional alkyl tail and an O-linked disaccharide composed of 3-α-epimycarose and 2-O-methyl-α-rhamnose. Incorporation of the disaccharide unit is preceded by a critical step involving hydroxylation by NocapM, a flavin monooxygenase. In this study, we employed biochemical, spectroscopic, and kinetic analyses to explore the substrate scope of NocapM. Our findings indicate that NocapM catalyzes hydroxylation of diverse aromatic substrates, although the observed coupling between NADPH oxidation and substrate hydroxylation varies widely from substrate to substrate. Our in-depth biochemical characterization of NocapM provides a solid foundation for future mechanistic studies of this enzyme as well as its utilization as a practical biocatalyst.