Identification of a magnesium-dependent NAD(P)(H)-binding domain in the nicotinoprotein methanol dehydrogenase from Bacillus methanolicus

HJ Hektor, H Kloosterman, L Dijkhuizen*

*Corresponding author for this work

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Abstract

The Bacillus methanolicus methanol dehydrogenase (MDH) is a decameric nicotinoprotein alcohol dehydrogenase (family III) with one Zn2+ ion, one or two Mg2+ ions, and a tightly bound cofactor NAD(H) per subunit. The Mg2+ ions are essential for binding of cofactor NAD(H) in MDH. A B. methanolicus activator protein strongly stimulates the relatively low coenzyme NAD(+)-dependent MDH activity, involving hydrolytic removal of the NMN(H) moiety of cofactor NAD(H) (Kloosterman, H., Vrijbloed, J. W., and Dijkhuizen, L. (2002) J. Biol. Chem. 277, 34785-34792). Members of family III of NAD(P)-dependent alcohol dehydrogenases contain three unique, conserved sequence motifs (domains A, B, and C). Domain C is thought to be involved in metal binding, whereas the functions of domains A and B are still unknown. This paper provides evidence that domain A constitutes (part of) a new magnesium-dependent NAD(P)(H)-binding domain. Site-directed mutants D100N and K103R lacked (most of the) bound cofactor NAD(H) and had lost all coenzyme NAD(+)-dependent MDH activity. Also mutants G95A and S97G were both impaired in cofactor NAD(H) binding but retained coenzyme NAD(+)-dependent MDH activity. Mutant G95A displayed a rather low MDH activity, whereas mutant S97G was insensitive to activator protein but displayed "fully activated" MDH reaction rates. The various roles of these amino acid residues in coenzyme and/or cofactor NAD(H) binding in MDH are discussed.

Original languageEnglish
Pages (from-to)46966-46973
Number of pages8
JournalThe Journal of Biological Chemistry
Volume277
Issue number49
DOIs
Publication statusPublished - 6-Dec-2002

Keywords

  • GLUCOSE-FRUCTOSE OXIDOREDUCTASE
  • ELECTRON-MICROSCOPIC ANALYSIS
  • ALCOHOL-DEHYDROGENASE
  • ZYMOMONAS-MOBILIS
  • SEQUENCE-ANALYSIS
  • ESCHERICHIA-COLI
  • CLOSTRIDIUM-ACETOBUTYLICUM
  • MOLECULAR CHARACTERIZATION
  • GENE-CLUSTER
  • THERMOTOLERANT BACILLUS

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