Exploring the biosynthetic capacity of TsrM, a B12‐dependent Radical SAM Enzyme Catalyzing Non‐radical Reactions

B12-dependent radical SAM enzymes are an emerging enzyme family with approximately 200,000 proteins. These enzymes have been shown to catalyze chemically challenging reactions such as methyl transfer to sp2 - and sp3 -hybridized carbon atoms. However, to date we have little information regarding their complex mechanisms and their biosynthetic potential. Here we show, using X-ray absorption spectroscopy, mutagenesis and synthetic probes that the vitamin B12-dependent radical SAM enzyme TsrM catalyzes not only C- but also N-methyl transfer reactions further expanding its synthetic versatility. We also demonstrate that TsrM has the unique ability to directly transfer a methyl group to the benzyl core of tryptophan, including the least reactive position C4. Collectively, our study supports that TsrM catalyzes non-radical reactions and establishes the usefulness of radical SAM enzymes for novel biosynthetic schemes including serial alkylation reactions at particularly inert C-H bonds.