Biochemical Characterization of a Dihydromethanopterin Reductase Involved in Tetrahydromethanopterin Biosynthesis in Methylobacterium extorquens AM1

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Abstract
During growth on one-carbon (C 1 ) compounds, the aerobic α-proteobacterium Methylobacterium extorquens AM1 synthesizes the tetrahydromethanopterin (H 4 MPT) derivative dephospho-H 4 MPT as a C 1 carrier in addition to tetrahydrofolate. The enzymes involved in dephospho-H 4 MPT biosynthesis have not been identified in bacteria. In archaea, the final step in the proposed pathway of H 4 MPT biosynthesis is the reduction of dihydromethanopterin (H 2 MPT) to H 4 MPT, a reaction analogous to the reaction of the bacterial dihydrofolate reductase. A gene encoding a dihydrofolate reductase homolog has previously been reported for M. extorquens and assigned as the putative H 2 MPT reductase gene ( dmrA ). In the present work, we describe the biochemical characterization of H 2 MPT reductase (DmrA), which is encoded by dmrA . The gene was expressed with a six-histidine tag in Escherichia coli , and the recombinant protein was purified by nickel affinity chromatography and gel filtration. Purified DmrA catalyzed the NAD(P)H-dependent reduction of H 2 MPT with a specific activity of 2.8 μmol of NADPH oxidized per min per mg of protein at 30°C and pH 5.3. Dihydrofolate was not a substrate for DmrA at the physiological pH of 6.8. While the existence of an H 2 MPT reductase has been proposed previously, this is the first biochemical evidence for such an enzyme in any organism, including archaea. Curiously, no DmrA homologs have been identified in the genomes of known methanogenic archaea, suggesting that bacteria and archaea produce two evolutionarily distinct forms of dihydromethanopterin reductase. This may be a consequence of different electron donors, NAD(P)H versus reduced F 420 , used, respectively, in bacteria and methanogenic archaea.

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