Structural Basis for Iterative Methylation by a Cobalamin-dependent Radical S-Adenosylmethionine Enzyme in...

Cystobactamids are non-ribosomal peptide natural products that function as DNA gyrase inhibitors, exhibiting significant antibacterial activity. They are isolated from Cystobacter sp. Cbv34 and contain various alkoxy groups on para-aminobenzoic acid moieties, which are believed to play a crucial role in antibacterial functions. The alkoxy groups are generated by iterative methylations on a methoxy group by the cobalamin (Cbl)-dependent radical S-adenosylmethionine (SAM) enzyme CysS. CysS catalyzes up to three methylations to give ethoxy, isopropoxy, sec-butoxy, and tert-butoxy groups. For each methylation, CysS uses a ping-pong mechanism in which two molecules of SAM are consumed. One SAM is used to methylate cob(I)alamin, while another generates a 5'-deoxyadenosyl 5'-radical to initiate substrate methylation. However, little is known about how the enzyme promotes both Cbl methylation and iterative substrate methylation, which occur by polar S_N2 and radical processes, respectively. Here, we report three X-ray crystal structures of a homolog of CysS from Corallococcus sp. CA054B. Two were determined in the presence of methoxy- and ethoxy-containing substrates, showing how CysS accommodates substrates and products during iterative methylation. The third structure, determined in the absence of a substrate, exhibits structural changes that reorient the SAM's conformation to allow for the methylation of cob(I)alamin.