The NADP-dependent methylene tetrahydromethanopterin dehydrogenase in Methylobacterium extorquens AM1.
An NADP-dependent methylene tetrahydromethanopterin (H4MPT) dehydrogenase has recently been proposed to be involved in formaldehyde oxidation to CO2 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 H4MPT 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 H4F) with NADP+. With methylene H4F as the substrate, however, the specific activity (26 U/mg) and the catalytic efficiency (Vmax/Km) were approximately 20-fold lower than with methylene H4MPT. Whereas the dehydrogenation of methylene H4MPT (E0 = -390 mV) with NADP+ (E0 = -320 mV) proceeded essentially irreversibly, the dehydrogenation of methylene H4F (E0 = -300 mV) was fully reversible. Comparison of the primary structure of the NADP-dependent dehydrogenase from M. extorquens AM1 with those of methylene H4F dehydrogenases from other bacteria and eucarya and with those of methylene H4MPT dehydrogenases from methanogenic archaea revealed only marginally significant similarity (<15%).
Max-Planck-Institut für terrestrische Mikrobiologie and Laboratorium für Mikrobiologie des Fachbereichs Biologie der Philipps-Universität, 35043 Marburg, Germany vorholt@mailer.uni-marburg.de
J A
JA
Vorholt
L
L
Chistoserdova
M E
ME
Lidstrom
R K
RK
Thauer
D000595:Amino Acid Sequence; D005557:Formaldehyde / Q000378:metabolism; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology*; D008954:Models, Biological; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism*; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000302:isolation & purification*; D011622:Pterins / Q000378:metabolism; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013763:Tetrahydrofolates / Q000032:analysis; D013816:Thermodynamics
C091917:N(5),N(10)-methylene-tetrahydromethanopterin; D011622:Pterins; D013763:Tetrahydrofolates; D005557:Formaldehyde; C030371:5,6,7,8-tetrahydrofolic acid; D009249:NADP; D000587:Oxidoreductases Acting on CH-NH Group Donors; C047126:methylenetetrahydromethanopterin dehydrogenase