Publications

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

  Hektor H.J., Kloosterman H., Dijkhuizen L.

  The Bacillus methanolicus methanol dehydrogenase (MDH) is a decameric nicotinoprotein alcohol dehydrogenase (family III) with one Zn(2+) ion, one or two Mg(2+) ions, and a tightly bound cofactor NAD(H) per subunit. The Mg(2+) ions are essential for binding of cofactor NAD(H) in MDH. A B. methanoli ... >> More

  The Bacillus methanolicus methanol dehydrogenase (MDH) is a decameric nicotinoprotein alcohol dehydrogenase (family III) with one Zn(2+) ion, one or two Mg(2+) ions, and a tightly bound cofactor NAD(H) per subunit. The Mg(2+) 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. << Less

  J. Biol. Chem. 277:46966-46973(2002) [PubMed] [EuropePMC]

• Purification and characterization of an activator protein for methanol dehydrogenase from thermotolerant Bacillus spp.

  Arfman N., Van Beeumen J., De Vries G.E., Harder W., Dijkhuizen L.

  All thermotolerant methanol-utilizing Bacillus spp. investigated by us possess a NAD-dependent methanol dehydrogenase (MDH) activity which is stimulated by a protein present in the soluble fraction of Bacillus sp. C1 cells. This activator protein was purified to homogeneity from Bacillus sp. C1 ce ... >> More

  All thermotolerant methanol-utilizing Bacillus spp. investigated by us possess a NAD-dependent methanol dehydrogenase (MDH) activity which is stimulated by a protein present in the soluble fraction of Bacillus sp. C1 cells. This activator protein was purified to homogeneity from Bacillus sp. C1 cells grown at a low dilution rate in a methanol-limited chemostat culture. The native activator protein (Mr = 50,000) is a dimer of Mr = 27,000 subunits. The N-terminal amino acid sequence revealed no significant similarity with any published sequences. Stimulation of MDH activity by the activator protein required the presence of Mg2+ ions. Plots of specific MDH activity versus activator protein concentration revealed Michaelis-Menten type kinetics. In the presence of activator protein, MDH displayed biphasic kinetics (v versus substrate concentration) toward C1-C4 primary alcohols and NAD. The data suggest that in the presence of activator protein plus Mg2+ ions, MDH possesses a high affinity active site for alcohols and NAD, in addition to an activator- and Mg2(+)-independent low affinity active site. The activation mechanism remains to be elucidated. << Less

  J. Biol. Chem. 266:3955-3960(1991) [PubMed] [EuropePMC]

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

  Arfman N., Hektor H.J., Bystrykh L.V., Govorukhina N.I., Dijkhuizen L., Frank J.

  Oxidation of C1-C4 primary alcohols in thermotolerant Bacillus methanolicus strains is catalyzed by an NAD-dependent methanol dehydrogenase (MDH), composed of ten identical 43,000-Mr subunits. Each MDH subunit contains a tightly, but non-covalently, bound NAD(H) molecule, in addition to 1 Zn2+ and ... >> More

  Oxidation of C1-C4 primary alcohols in thermotolerant Bacillus methanolicus strains is catalyzed by an NAD-dependent methanol dehydrogenase (MDH), composed of ten identical 43,000-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 50,000-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. << Less

  Eur J Biochem 244:426-433(1997) [PubMed] [EuropePMC]

• Methanol metabolism in thermotolerant methylotrophic Bacillus strains involving a novel catabolic NAD-dependent methanol dehydrogenase as a key enzyme.

  Arfman N., Watling E.M., Clement W., van Oosterwijk R.J., de Vries G.E., Harder W., Attwood M.M., Dijkhuizen L.

  The enzymology of methanol utilization in thermotolerant methylotrophic Bacillus strains was investigated. In all strains an immunologically related NAD-dependent methanol dehydrogenase was involved in the initial oxidation of methanol. In cells of Bacillus sp. C1 grown under methanol-limiting con ... >> More

  The enzymology of methanol utilization in thermotolerant methylotrophic Bacillus strains was investigated. In all strains an immunologically related NAD-dependent methanol dehydrogenase was involved in the initial oxidation of methanol. In cells of Bacillus sp. C1 grown under methanol-limiting conditions this enzyme constituted a high percentage of total soluble protein. The methanol dehydrogenase from this organism was purified to homogeneity and characterized. In cell-free extracts the enzyme displayed biphasic kinetics towards methanol, with apparent Km values of 3.8 and 166 mM. Carbon assimilation was by way of the fructose-1,6-bisphosphate aldolase cleavage and transketolase/transaldolase rearrangement variant of the RuMP cycle of formaldehyde fixation. The key enzymes of the RuMP cycle, hexulose-6-phosphate synthase (HPS) and hexulose-6-phosphate isomerase (HPI), were present at very high levels of activity. Failure of whole cells to oxidize formate, and the absence of formaldehyde- and formate dehydrogenases indicated the operation of a non-linear oxidation sequence for formaldehyde via HPS. A comparison of the levels of methanol dehydrogenase and HPS in cells of Bacillus sp. C1 grown on methanol and glucose suggested that the synthesis of these enzymes is not under coordinate control. << Less

  Arch. Microbiol. 152:280-288(1989) [PubMed] [EuropePMC]

• Molecular, biochemical, and functional characterization of a Nudix hydrolase protein that stimulates the activity of a nicotinoprotein alcohol dehydrogenase.

  Kloosterman H., Vrijbloed J.W., Dijkhuizen L.

  The cytoplasmic coenzyme NAD(+)-dependent alcohol (methanol) dehydrogenase (MDH) employed by Bacillus methanolicus during growth on C(1)-C(4) primary alcohols is a decameric protein with 1 Zn(2+)-ion and 1-2 Mg(2+)-ions plus a tightly bound NAD(H) cofactor per subunit (a nicotinoprotein). Mg(2+)-i ... >> More

  The cytoplasmic coenzyme NAD(+)-dependent alcohol (methanol) dehydrogenase (MDH) employed by Bacillus methanolicus during growth on C(1)-C(4) primary alcohols is a decameric protein with 1 Zn(2+)-ion and 1-2 Mg(2+)-ions plus a tightly bound NAD(H) cofactor per subunit (a nicotinoprotein). Mg(2+)-ions are essential for binding of NAD(H) cofactor in MDH protein expressed in Escherichia coli. The low coenzyme NAD(+)-dependent activity of MDH with C(1)-C(4) primary alcohols is strongly stimulated by a second B. methanolicus protein (ACT), provided that MDH contains NAD(H) cofactor and Mg(2+)-ions are present in the assay mixture. Characterization of the act gene revealed the presence of the highly conserved amino acid sequence motif typical of Nudix hydrolase proteins in the deduced ACT amino acid sequence. The act gene was successfully expressed in E. coli allowing purification and characterization of active ACT protein. MDH activation by ACT involved hydrolytic removal of the nicotinamide mononucleotide NMN(H) moiety of the NAD(H) cofactor of MDH, changing its Ping-Pong type of reaction mechanism into a ternary complex reaction mechanism. Increased cellular NADH/NAD(+) ratios may reduce the ACT-mediated activation of MDH, thus preventing accumulation of toxic aldehydes. This represents a novel mechanism for alcohol dehydrogenase activity regulation. << Less

  J. Biol. Chem. 277:34785-34792(2002) [PubMed] [EuropePMC]