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Abstract:
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 K-m for
methylene H4MPT was 50 mu M with NAD(+) (V-max = 1100 U.mg(-1)) and 100
mu M with NADP(+) (V-max = 950 U.mg(-1)). The K-m value for NAD(+) was
200 mu M and for NADP(+) 20 mu M. In contrast to MtdA, MtdB could not
catalyze the dehydrogenation of methylene tetrahydrofolate. Via the
N-terminal aminoacid 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.
