Purification and characterisation of TOL plasmid-encoded benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase of Pseudomonas putida.
Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase, two enzymes of the xylene degradative pathway encoded by the plasmid TOL of a Gram-negative bacterium Pseudomonas putida, were purified and characterized. Benzyl alcohol dehydrogenase catalyses the oxidation of benzyl alcohol to benzaldehyde with the concomitant reduction of NAD+; the reaction is reversible. Benzaldehyde dehydrogenase catalyses the oxidation of benzaldehyde to benzoic acid with the concomitant reduction of NAD+; the reaction is irreversible. Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase also catalyse the oxidation of many substituted benzyl alcohols and benzaldehydes, respectively, though they were not capable of oxidizing aliphatic alcohols and aldehydes. The apparent Km value of benzyl alcohol dehydrogenase for benzyl alcohol was 220 microM, while that of benzaldehyde dehydrogenase for benzaldehyde was 460 microM. Neither enzyme contained a prosthetic group such as FAD or FMN, and both enzymes were inactivated by SH-blocking agents such as N-ethylmaleimide. Both enzymes were dimers of identical subunits; the monomer of benzyl alcohol dehydrogenase has a mass of 42 kDa whereas that of the monomer of benzaldehyde dehydrogenase was 57 kDa. Both enzymes transfer hydride to the pro-R side of the prochiral C4 of the pyridine ring of NAD+.
Département de Biochimie Médicale, Centre Médical Universitaire, Genève, Switzerland.
J P
JP
Shaw
S
S
Harayama
D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D000445:Aldehyde Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005033:Ethylmaleimide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D010957:Plasmids; D011549:Pseudomonas / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
D046911:Macromolecular Substances; D009243:NAD; D000429:Alcohol Oxidoreductases; C027016:benzyl alcohol dehydrogenase; D000445:Aldehyde Oxidoreductases; C027015:benzaldehyde dehydrogenase (NAD+); D005033:Ethylmaleimide