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Abstract:
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 (V-max/K-m) were approximately 20-fold
lower than with methylene H4MPT, Whereas the dehydrogenation of
methylene H4MPT (E-0 = -390 mV) with NADP(+) (E-0 = -320 mV) proceeded
essentially irreversibly, the dehydrogenation of methylene H4F (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 H4F dehydrogenases from other bacteria and eucarya and with
those of methylene H4MPT dehydrogenases from methanogenic archaea
revealed only marginally significant similarity (<15%).
