title: Purification and characterization of an activator protein for methanol dehydrogenase from thermotolerant Bacillus spp.
abstract: 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.
authors.affiliation: Department of Microbiology, University of Groningen, Haren, The Netherlands.
authors.forename: N
authors.initials: N
authors.lastname: Arfman
authors.forename: J
authors.initials: J
authors.lastname: Van Beeumen
authors.forename: G E
authors.initials: GE
authors.lastname: De Vries
authors.forename: W
authors.initials: W
authors.lastname: Harder
authors.forename: L
authors.initials: L
authors.lastname: Dijkhuizen
mesh_terms: D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000438:Alcohols / Q000737:chemistry; D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006358:Hot Temperature; D007700:Kinetics; D010084:Oxidation-Reduction
chemical_list: D000438:Alcohols; D001426:Bacterial Proteins; D000429:Alcohol Oxidoreductases; C019641:alcohol dehydrogenase (acceptor)