A Cobalamin‐Dependent Radical SAM Enzyme Catalyzes the Unique C(α)‐Methylation of Glutamine in Methyl‐Coenzyme M Reductase

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.