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.

S-Adenosylmethionine (SAM) is an essential and versatile cofactor in nature. SAM-dependent enzymes, such as conventional methyltransferases (MTs), amino(carboxy)propyl transferases, and radical SAM enzymes, are of great interest as biocatalytic tools for chemical synthesis and the pharmaceutical industry. The use of SAM analogues adds to the diversity of reaction types and products. While SAM regeneration protocols for conventional MTs are available, in vitro SAM regeneration for polyamine-forming enzymes and radical SAM enzymes has not been solved yet. Here, we report a biomimetic cofactor regeneration cycle for three important types of SAM dependent enzymes featuring adenine as a central intermediate. SAM is regenerated from its main degradation products, S-adenosylhomocysteine, 5′ methylthioadenosine, and 5′-deoxyadenosine. We further show the chemical diversification of the system using SAM analogues such as S-adenosylethionine for MT reactions. Interestingly, the enzyme cascades also enable the transfer of an ethyl group with a cobalamin-dependent radical SAM MT. The possibility to regenerate SAM (analogues) for biocatalytic use with a broad range of enzymes will be a starting point for further application of the diverse chemistry accessible by SAM-based catalysis, as well as provide new options for mechanistic studies.

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.