Methyl-coenzyme M reductase, which is responsible for the production of the greenhouse gas methane during biological methane formation, carries several unique posttranslational amino acid modifications, including a 2-(S)methylglutamine. The enzyme responsible for the C α -methylation of this glutamine is not known. Herein, we identify and characterize a cobalamin-dependent radical SAM enzyme as the glutamine C-methyltransferase. The recombinant protein from Methanoculleus thermophilus binds cobalamin in a base-off, His-off conformation and contains a single [4Fe-4S] cluster. The cobalamin cofactor cycles between the methyl-cob(III)alamin, cob(II)alamin and cob(I)alamin states during catalysis and produces methylated substrate, 5'-deoxyadenosine and S-adenosyl-L-homocysteine in a 1 : 1 : 1 ratio. The newly identified glutamine C-methyltransferase belongs to the class B radical SAM methyltransferases known to catalyze challenging methylation reactions of sp 3 -hybridized carbon atoms.

S‐Adenosylmethionine (SAM) is an enzyme cofactor involved in methylation, aminopropyl transfer, and radical reactions. This versatility renders SAM‐dependent enzymes of great interest in biocatalysis. The usage of SAM analogues adds to this diversity. However, high cost and instability of the cofactor impedes the investigation and usage of these enzymes. While SAM regeneration protocols from the methyltransferase (MT) byproduct S‐adenosylhomocysteine are available, aminopropyl transferases and radical SAM enzymes are not covered. Here, we report a set of efficient one‐pot systems to supply or regenerate SAM and SAM analogues for all three enzyme classes. The systems’ flexibility is showcased by the transfer of an ethyl group with a cobalamin‐dependent radical SAM MT using S‐adenosylethionine as a cofactor. This shows the potential of SAM (analogue) supply and regeneration for the application of diverse chemistry, as well as for mechanistic studies using cofactor analogues.

Methyl-coenzyme M reductase, which is responsible for the production of the greenhouse gas methane during biological methane formation, carries several unique posttranslational amino acid modifications, including a 2-(S)methylglutamine. The enzyme responsible for the C α -methylation of this glutamine is not known. Herein, we identify and characterize a cobalamin-dependent radical SAM enzyme as the glutamine C-methyltransferase. The recombinant protein from Methanoculleus thermophilus binds cobalamin in a base-off, His-off conformation and contains a single [4Fe-4S] cluster. The cobalamin cofactor cycles between the methyl-cob(III)alamin, cob(II)alamin and cob(I)alamin states during catalysis and produces methylated substrate, 5'-deoxyadenosine and S-adenosyl-L-homocysteine in a 1 : 1 : 1 ratio. The newly identified glutamine C-methyltransferase belongs to the class B radical SAM methyltransferases known to catalyze challenging methylation reactions of sp 3 -hybridized carbon atoms.