The NADP-Dependent Methylene Tetrahydromethanopterin Dehydrogenase in Methylobacterium extorquens AM1

Abstract

ABSTRACT 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%).