A multifunctional fermentative alcohol dehydrogenase from the strict aerobe Alcaligenes eutrophus: purification and properties.
A NAD (P)-linked alcohol dehydrogenase was isolated from the soluble extract of the strictly respiratory bacterium Alcaligenes eutrophus N9A. Derepression of the formation of this enzyme occurs only in cells incubated under conditions of restricted oxygen supply for prolonged times. The purification procedure included precipitation by cetyltrimethylammonium bromide and ammonium sulfate and subsequent chromatography on DEAE-Sephacel, Cibacron blue F3G-A Sepharose and thiol-Sepharose. The procedure resulted in a 120-fold purification of a multifunctional alcohol dehydrogenase exhibiting dehydrogenase activities for 2,3-butanediol, ethanol and acetaldehyde and reductase activities for diacetyl, acetoin and acetaldehyde. During purification the ratio between 2,3-butanediol dehydrogenase and ethanol dehydrogenase activity remained nearly constant. Recovering about 20% of the initial 2,3-butanediol dehydrogenase activity, the specific activity of the final preparation was 70.0 U X mg protein-1 (2,3-butanediol oxidation) and 2.8 U X mg protein-1 (ethanol oxidation). The alcohol dehydrogenase is a tetramer of a relative molecular mass of 156000 consisting of four equal subunits. The determination of the Km values for different substrates and coenzymes as well as the determination of the pH optima for the reactions catalyzed resulted in values which were in good agreement with the fermentative function of this enzyme. The alcohol dehydrogenase catalyzed the NAD (P)-dependent dismutation of acetaldehyde to acetate and ethanol. This reaction was studied in detail, and its possible involvement in acetate formation is discussed. Among various compounds tested for affecting enzyme activity only NAD, NADP, AMP, ADP, acetate and 2-mercaptoethanol exhibited significant effects.
D000421:Alcaligenes / Q000201:enzymology*; D000426:Alcohol Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D000445:Aldehyde Oxidoreductases / Q000378:metabolism; D002845:Chromatography / Q000379:methods; D003067:Coenzymes / Q000378:metabolism; D003653:Decarboxylation; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008623:Mercaptoethanol / Q000494:pharmacology; D008970:Molecular Weight; D010084:Oxidation-Reduction; D013379:Substrate Specificity
D003067:Coenzymes; D008623:Mercaptoethanol; D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase; C019801:butanediol dehydrogenase; D000445:Aldehyde Oxidoreductases; C018699:aldehyde dehydrogenase (NAD(P)+)