Biochemical Characterization of the Radical SAM Methylase Involved in Tetrahydromethanopterin Biosynthesis in Methanogenic Archaea

Tetrahydromethanopterin (H4MPT) is a tetrahydrofolate (H4F) analog that serves as a one-carbon carrier cofactor in the methanogenesis pathway in methanogenic microorganisms. One major structural feature that distinguishes H4MPT from H4F and other pterin-containing biomolecules is the presence of methyl groups at the C7 and C9 positions. Although the biosynthesis of H4 MPT has been relatively well-defined, the enzyme responsible for the methylation reactions at the unreactive carbon centers has remained unclear. Our previous work identified the radical S-adenosyl-L-methionine (SAM) methylase, MjMptM, from Methanocaldococcus jannaschii that likely catalyzes the addition of both methyl groups during H4 MPT biosynthesis. Our current data indicate that MjMptM does not utilize SAM as the methyl group donor and instead uses methylenetetrahydrofolate for this purpose, thus making MjMptM the founding member of "Class D" radical SAM methylases. Here, we describe our recent progress towards the biochemical and spectroscopic characterization of MjMptM. We demonstrated that the enzyme catalyzes two methylation reactions to produce a dimethylated folate species using methylenetetrahydrofolate as the C1 source for the in vitro methylation reactions and dihydrofolate as the in vitrosubstrate. Site-directed mutagenesis and UV-Vis spectroscopic analysis indicate that MjMptM harbors at least two [4Fe-4S] clusters which reside in the two canonical CX3 CX2 C radical SAM motifs in the N-terminus. This result was further confirmed by EPR spectroscopy, which shows the binding of at least two [4Fe-4S] clusters with unique signals. Only one of the [4Fe-4S] clusters catalyzes the reductive cleavage of SAM to produce 5'deoxyadenosine, the key first step in radical SAM enzyme catalysis, and thus the most N-terminal auxiliary cluster is likely involved in electron transfer. Current work is focused on elucidating the details of the novel methylation reaction mechanism.