P24: Biochemical and structural analysis of MycF, an O-methyltransferase from the mycinamicin biosynthetic pathway

The natural product mycinamicin, produced by the rare actinomycete Micromonospora griseorubida, is a macrolide antibiotic demonstrating potent antibacterial activity against a number of Gram-positive bacteria, including select antibiotic-resistant human pathogens. The macrolide is composed of a 16-membered macrolactone and two sugars, desosamine and mycinose located at the C-5 and C-21 positions. Previous reports indicated that the mycinose sugar is synthesized through the sequential methylation of 6-deoxyallose by S-adenosyl-L-methionine- and metal-dependent O-methyltransferases MycE and MycF. MycE catalyzes methylation of 6-deoxyallose at the C-2’ hydroxyl group, producing javose in mycinamicin III, and MycF methylates the C-3’ hydroxyl group of the same sugar to generate mycinose in mycianamicin IV.In this study, we report the biochemical and structural analysis of MycF, a substrate promiscuious methyltransferase capable of methylating a javose sugar on exogenous substrates derived from tylosin biosynthetic pathway. To confirm the in vitro enzymatic conversions, enzyme-substrate co-crystal structures were determined for MycF bound to the S-adenosyl-L-homocysteine and magnesium, with the natural substrate, mycinamicin III, and unnatural substrate, macrocin. The interactions between MycF and macrolactones highlight the potential of this flexible O-methyltransferase as a biocatalyst for the generation of novel bioactives. Finally, our results provide valuable insight into the enzymatic sugar modifications that occur during macrolide biosynthesis.