Purification, crystallisation and characterization of quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa.
Pseudomonas aeruginosa ATCC 17933 when grown on ethanol produces high levels of a quinoprotein ethanol dehydrogenase, which amounts to 7% of the soluble protein. The enzyme has been purified to homogeneity and it crystallizes readily in the presence of polyethylene glycol 1550 or 6000. The ethanol dehydrogenase (Km(ethanol) = 14 microM) resembles the dye-dependent quinoprotein methanol dehydrogenases of methylotrophic bacteria, but has a low affinity for methanol (Km (methanol) = 94mM). In addition the enzyme oxidizes secondary alcohols. With its catalytic properties the ethanol dehydrogenase is similar to the enzyme isolated from P. aeruginosa LMD 80.53 (Groen, B., Frank, J. Jzn. & Duine, J.A. (1984) Biochem. J. 223, 921-924). In contrast to this enzyme from P. aeruginosa LMD 80.53, which is a monomer, the ethanol dehydrogenase isolated from P. aeruginosa ATCC 17933 is a dimer of identical subunits of relative molecular mass 60,000. The N-terminal amino acid is lysine. Inactivation with cyclopropanone ethylhemiketal reveals one molecule of pyrroloquinoline quinone per subunit. As shown by active enzyme sedimentation, the dimer is the enzymatically active form.
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Institut für Mikrobiologie der Universität Hohenheim.
Görisch
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D000429:Alcohol Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003460:Crystallization; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D011549:Pseudomonas / Q000201:enzymology; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013045:Species Specificity; D013379:Substrate Specificity
D000596:Amino Acids; D046911:Macromolecular Substances; D000429:Alcohol Oxidoreductases; C019641:alcohol dehydrogenase (acceptor)