Properties of an NAD(H)-containing methanol dehydrogenase and its activator protein from Bacillus methanolicus

Nico Arfman, Harm J. Hektor, Leonid V. Bystrykh, Natalya I. Govorukhina, Lubbert Dijkhuizen, Johannes Frank

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Abstract

Oxidation of C1-C4 primary alcohols in thermotolerant Bacillus methanolicus strains is catalyzed by an NAD-dependent methanol dehydrogenase (MDH), composed of ten identical 43000-Mr subunits. Each MDH subunit contains a tightly, but non-covalently, bound NAD(H) molecule, in addition to 1 Zn2+ and 1-2 Mg2+ ions. The NAD(H) cofactor is oxidized and reduced by formaldehyde and methanol, respectively, while it remains bound to the enzyme. Incubation of MDH with methanol and exogenous NAD (coenzyme) results in reduction of this NAD coenzyme. Both NAD species are not exchanged during catalysis. NAD thus plays two different and important roles in the MDH-catalyzed reaction, with the bound NAD cofactor acting as primary electron acceptor and the NAD coenzyme being responsible for reoxidation of the reduced cofactor. MDH obeys a ping-pong type reaction mechanism, which is consistent with such a temporary parking of reducing equivalents at the MDH-bound cofactor. Spectral studies show that, in the presence of exogenous NAD and Mg2+ ions, MDH interacts with a previously identified 50000-Mr activator protein. The activator protein appears to facilitate the oxidation of the reduced NADH cofactor of MDH, which results in a strongly increased turnover rate of MDH.
Original languageEnglish
Pages (from-to)426-433
Number of pages8
JournalEuropean Journal of Biochemistry
Volume244
Issue number2
DOIs
Publication statusPublished - 1-Mar-1997

Keywords

  • methanol dehydrogenase
  • NAD
  • activator protein
  • Bacillus methanolicus
  • ELECTRON-MICROSCOPIC ANALYSIS
  • LIVER ALCOHOL-DEHYDROGENASE
  • GRAM-POSITIVE BACTERIA
  • ZYMOMONAS-MOBILIS
  • AMYCOLATOPSIS-METHANOLICA
  • SACCHAROMYCES-CEREVISIAE
  • THERMOTOLERANT BACILLUS
  • FORMALDEHYDE DISMUTASE
  • OXIDOREDUCTASE
  • GENE

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