The NADP-Dependent Methylene Tetrahydromethanopterin Dehydrogenase in Methylobacterium extorquens AM1

An NADP-dependent methylene tetrahydromethanopterin (H(4)MPT) dehydrogenase has recently been proposed to be involved in formaldehyde oxidation to CO(2) in Methylobacterium extorquens AM1. We report here on the purification of this novel enzyme to apparent homogeneity. Via the N-terminal amino acid sequence, it was identified to be the mtdA gene product. The purified enzyme catalyzed the dehydrogenation of methylene H(4)MPT with NADP(+) rather than with NAD(+), with a specific activity of approximately 400 U/mg of protein. It also catalyzed the dehydrogenation of methylene tetrahydrofolate (methylene H(4)F) with NADP(+). With methylene H(4)F as the substrate, however, the specific activity (26 U/mg) and the catalytic efficiency (V(max)/K(m)) were approximately 20-fold lower than with methylene H(4)MPT. Whereas the dehydrogenation of methylene H(4)MPT (E(0) = −390 mV) with NADP(+) (E(0) = −320 mV) proceeded essentially irreversibly, the dehydrogenation of methylene H(4)F (E(0) = −300 mV) was fully reversible. Comparison of the primary structure of the NADP-dependent dehydrogenase from M. extorquens AM1 with those of methylene H(4)F dehydrogenases from other bacteria and eucarya and with those of methylene H(4)MPT dehydrogenases from methanogenic archaea revealed only marginally significant similarity (<15%).