A Cobalamin‐Dependent Radical SAM Enzyme Catalyzes the Unique C_α‐Methylation of Glutamine in Methyl‐Coenzyme M Reductase

Abstract

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.

Versatile enzymology and heterogeneous phenotypes in cobalamin complementation type C disease

“…It is shown that cob(II)alamin forms a relatively stable six-coordinated complex with sulfur dioxide anion radical SO 2 − having strong reducing properties. Indeed, dithionite (sulfur dioxide anion radical) produces the catalytically incompetent cob(II)alamin ( Allen and Wang, 2014 ; Gagsteiger et al., 2022 ). Insights on the importance of the upper axial ligand in cobalamin structure and reactivity can be derived from a comparison of the structure of cob(II)alamin-SO 2 − with the other important complex – nitrosylcobalamin NOCbl ( Hannibal et al., 2007 ; Hassanin et al., 2009b ), which is readily formed when cob(II)alamin reacts with nitric oxide.…”

Biomimetic S-Adenosylmethionine Regeneration Starting from Different Byproducts Enables Biocatalytic Alkylation with Radical SAM Enzymes

“…5a). [52] As both, DOA and SAH, are substrates for MTAN [34] , the regeneration system should be capable of cleaving both byproducts (SAH and DOA) and regenerating two equivalents of SAM from adenine per methylation reaction.…”

“…[52] A 24-mer peptide fragment of methyl-coenzyme M reductase was used as the substrate as described previously. [52] Analysis of the linear cascade consisting of MAT, QCMT, and MTAN by liquid chromatography mass spectrometry (LC-MS) revealed full conversion after 20 h (Fig. S5a).…”

“…Samples for the analysis of cobalamin bound to the enzyme were prepared as previously described. [52] Assays for investigating the SAM regeneration system with QCMT were carried out in 50 mM…”

“…Fig.5SAM regeneration for class B radical SAM MTs. a) QCMT methylates C2 of a glutamine residue within a peptide chain (orange) under retention of stereochemistry [52]. Two SAM molecules are utilised, one for radical generation, the other for cobalamin methylation.DOA and SAH are formed as byproducts, both can be regenerated to SAM (yellow circle).…”