Characterization of a second methylene tetrahydromethanopterin dehydrogenase from Methylobacterium extorquens AM1.
Cell extracts of Methylobacterium extorquens AM1 were recently found to catalyze the dehydrogenation of methylene tetrahydromethanopterin (methylene H4MPT) with NAD+ and NADP+. The purification of a 32-kDa NADP-specific methylene H4MPT dehydrogenase (MtdA) was described already. Here we report on the characterization of a second methylene H4MPT dehydrogenase (MtdB) from this aerobic alpha-proteobacterium. Purified MtdB with an apparent molecular mass of 32 kDa was shown to catalyze the oxidation of methylene H4MPT to methenyl H4MPT with NAD+ and NADP+ via a ternary complex catalytic mechanism. The Km for methylene H4MPT was 50 microM with NAD+ (Vmax = 1100 U x mg(-1) and 100 microM with NADP+ (Vmax = 950 U x mg(-1). The Km value for NAD+ was 200 microM and for NADP+ 20 microM. In contrast to MtdA, MtdB could not catalyze the dehydrogenation of methylene tetrahydrofolate. Via the N-terminal amino-acid sequence, the MtdB encoding gene was identified to be orfX located in a cluster of genes whose translated products show high sequence identities to enzymes previously found only in methanogenic and sulfate reducing archaea. Despite its location, MtdB did not show sequence similarity to archaeal enzymes. The highest similarity was to MtdA, whose encoding gene is located outside of the archaeal island. Mutants defective in MtdB were unable to grow on methanol and showed a pronounced sensitivity towards formaldehyde. On the basis of the mutant phenotype and of the kinetic properties, possible functions of MtdB and MtdA are discussed. We also report that both MtdB and MtdA can be heterologously overproduced in Escherichia coli making these two enzymes readily available for structural analysis.
Hagemeier
C H
CH
Max-Planck-Institut für terrestrische Mikrobiologie, Marburg, Germany.
Chistoserdova
L
L
Lidstrom
M E
ME
Thauer
R K
RK
Vorholt
J A
JA
D000595:Amino Acid Sequence; D002455:Cell Division / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005557:Formaldehyde / Q000494:pharmacology; D007700:Kinetics; D000432:Methanol / Q000378:metabolism; D020601:Methylobacterium extorquens / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D009243:NAD; D005557:Formaldehyde; D009249:NADP; D000587:Oxidoreductases Acting on CH-NH Group Donors; C047126:methylenetetrahydromethanopterin dehydrogenase; D000432:Methanol