Radical S -adenosylmethionine (SAM) enzymes catalyze an impressive variety of difficult biochemical reactions in various pathways across all domains of life. These metalloenzymes employ a reduced [4Fe-4S] cluster and SAM to generate a highly reactive 5′-deoxyadenosyl radical that is capable of initiating catalysis on otherwise unreactive substrates.
Radical S‐adenosyl methionine (SAM) enzymes utilize SAM and a [4Fe‐4S] cluster to catalyze complex radical reactions throughout various biochemical processes. Methylases are one of the largest subgroups of radical SAM enzymes, which add a methyl group to otherwise unreactive substrates. Our previous work identified the radical SAM methylase, MjMptM, from Methanocaldococcus jannaschii, which likely catalyzes the addition of methyl groups to the C7 and C9 positions of tetrahydromethanopterin, a central C1‐carrier cofactor in methanogenic archaea. MjMptM is a unique methylase since it contains multiple [4Fe‐4S] clusters and our current experimental evidence indicates that it does not utilize SAM as the methyl group donor. Here, we heterologously expressed and purified MjMptM for in vitro enzymatic and spectroscopic studies. We demonstrated that the enzyme catalyzes two methylation reactions to produce a dimethylated folate species likely using methylenetetrahydrofolate as the C1 source for the methylation reactions. Site‐directed mutagenesis and UV‐Vis spectroscopic analysis indicate that MjMpt harbors three [4Fe‐4S] clusters, two of which reside in the two canonical CX3CX2C radical SAM motifs in the N‐terminus. Interestingly, our results indicate that both of the [4Fe‐4S] clusters within radical SAM motifs catalyze the reductive cleavage of SAM to produce 5’deoxyadenosine, the key first step in radical SAM enzyme catalysis. The exact location and function of the third auxiliary cluster is yet to be determined, but we propose it is bound by conserved cysteine residues in the C‐terminus. Current work is focused on understanding the role of this auxiliary cluster as well as details of the methylation reaction mechanism.
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 H4MPT 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 H4MPT 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 CX3CX2C 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.
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