This group of enzymes currently only contains one family, which has methyltransferase activity that has unique features from other identified methyltransferases. Here, the methyl donor is thought to be folate. Thus, this subgroup has been termed the Class D Methyltransferases.
Allen KD, Xu H, White RH
Identification of a unique radical SAM methylase likely involved in methanopterin biosynthesis in Methanocaldococcus jannaschii
▸ Abstract
Methanopterin (MPT) and its analogs are coenzymes required for methanogenesis and methylotrophy in specialized microorganisms. The methyl groups at C-7 and C-9 of the pterin ring distinguish MPT from all other pterin-containing natural products. However, the enzyme(s) responsible for addition of these methyl groups has yet to be identified. Here we demonstrate that a putative radical S-adenosyl-L-methionine (SAM) enzyme superfamily member encoded by the mj0619 gene in the methanogen Methanocaldococcus jannaschii is likely this missing methylase. When MJ0619 was heterologously expressed in Escherichia coli, various methylated pterins were detected, consistent with MJ0619 catalyzing methylation at C-7 and C-9 of 7,8-dihydro-6-hydroxymethylpterin, a common intermediate in both folate and MPT biosynthesis. Site-directed mutagenesis of Cys77 present in the first of two canonical radical SAM CX3CX2C motifs present in MJ0619 did not inhibit C-7 methylation while mutation of Cys102, found in the other radical SAM amino acid motif, resulted in loss of C-7 methylation, suggesting that the first motif is likely involved in C-9 methylation while the second motif is required for C-7 methylation. Further experiments demonstrated that the C-7 methyl group is not derived from methionine and methylation does not require cobalamin. When the E. coli expressing MJ0619 was grown in minimal media with deuterium-labeled acetate as the sole carbon source, the resulting methyl group on the pterin was predominantly labeled with three deuteriums. Based on these results, we propose that this archaeal radical SAM methylase employs a previously uncharacterized mechanism for methylation, using methylenetetrahydrofolate as a methyl group donor.
Mechanistic Diversity of Radical SAM-Dependent Methylation
▸ Abstract
Radical S-adenosylmethionine (SAM) enzymes use the oxidizing power of a 5'-deoxyadenosyl 5'-radical to initiate an amazing array of transformations, usually through the abstraction of a target substrate hydrogen atom. A common reaction of radical SAM (RS) enzymes is the methylation of unactivated carbon or phosphorous atoms found in numerous primary and secondary metabolites, as well as in proteins, sugars, lipids, and RNA. However, neither the chemical mechanisms by which these unactivated atoms obtain methyl groups nor the actual methyl donors are conserved. In fact, RS methylases have been grouped into three classes based on protein architecture, cofactor requirement, and predicted mechanism of catalysis. Class A methylases use two cysteine residues to methylate sp2-hybridized carbon centers. Class B methylases require a cobalamin cofactor to methylate both sp2- and sp3-hybridized carbon centers as well as phosphinate phosphorous atoms. Class C methylases share significant sequence homology with the RS enzyme, HemN, and may bind two SAM molecules simultaneously to methylate sp2-hybridized carbon centers. Lastly, we describe a new class of recently discovered RS methylases. These Class D methylases, unlike Class A, B, and C enzymes, which use SAM as the source of the donated methyl carbon, are proposed to methylate sp2-hybridized carbon centers using methylenetetrahydrofolate as the source of the appended methyl carbon.
