Iterative Methylations Resulting in the Biosynthesis of the t-Butyl Group Catalyzed by a B-Dependent Radical SAM Enzyme in Cystobactamid Biosynthesis.

B-dependent radical SAM enzymes that can perform methylations on sp carbon centers are important for functional diversity and regulation of biological activity in several nonribosomal peptides. Detailed studies on these enzymes are hindered by the complexity of the substrates and low levels of expression of active enzymes. CysS can catalyze iterative methylations of a methoxybenzene moiety during the biosynthesis of the cystobactamids. Here, we describe the overexpression, purification, substrate identification, and mechanism of this enzyme.