Mechanism of C-C Bond Forming Radical SAM Enzymes in Antifungal Natural Product and Molybdenum Cofactor Biosynthesis

Abstract

Naturally occurring small molecules are often characterized by their unique and complex structures critical for their biological functions. Many of these natural product biosynthetic pathways involve radical S-adenosylmethionine (SAM) enzymes. These enzymes form one of the largest enzyme superfamilies, and are characterized by their ability to catalyze the reductive cleavage of SAM using oxygen sensitive 4Fe-4S clusters. Generally, these reactions generate a transient 5′-dA• which abstracts an H-atom from the substrate and initiate free-radical mediated reactions. In the past five years, many radical SAM enzymes have been found to catalyze key C-C bond forming steps in various biological processes, but their mechanisms are largely uncharacterized. This dissertation focuses the functional and mechanistic characterization of two distinct C-C bond forming radical SAM enzymes. In the first part of the dissertation, I describe my successful identification and characterization of PolH that catalyzes a key step in the biosynthesis of a group of antifungal peptidyl nucleosides. The mechanistic characterization of PolH provided the first evidence for the mechanism of radical quenching in the reactions catalyzed by C-C bond forming radical SAM enzymes. In the second part, I report the first characterization of 5’-dA-associated radicals in a MoaA variant with mutations that cause human molybdenum cofactor deficiency. The results provided the first experimental evidence for the mechanism of radical quenching in MoaA. The study also provides important insights into the mechanism of SAM cleavage and the reactivity of 5’-dA• that may be generally applicable to other radical SAM enzymes. My studies on these two radical SAM enzymes provided the first mechanistic insights into the free radical mediated C-C bond formation in radical SAM enzymes.