A NEW ALCOHOL-DEHYDROGENASE WITH UNIQUE STEREOSPECIFICITY FROM PSEUDOMONAS SP

A new nicotinamide cofactor-dependent alcohol dehydrogenase from Pseudomonas strain SBD6 (PADH) was isolated and purified 150-fold to homogeneity using a combination of salt precipitation, anion-exchange chromatography, gel filtration chromatography, and dye matrix chromatography. Approximately 10 mg of pure enzyme can be obtained from 10 g of wet cells. The enzyme has four subunits with a total molecular weight of 162,000. Incubation with the metal chelators 1,10-phenanthroline, 2-aminoethanethiol, hydroxyquinolinesulfonic acid, N-ethylmaleimide, and potassium cyanide result in complete loss of activity. The enzyme is very stable (t 1 2 ∼ 7 days at pH 7 and 25°C in the absence of 2-propanol and ∼ 18 days in the presence of 10% 2-propanol, v/v) and possesses a broad substrate specificity with transfer of the pro-(R) hydride from NADH to the si face of carbonyl substrates to give (R)-alcohols in high enantiomeric excess, a stereochemical process different from that of other known alcohol dehydrogenases. Synthetic scale reductions are facilitated with 2-propanol as a hydride source for the regeneration of NADH. The kinetic mechanism is ordered bi-bi with the cofactor binding first. Based on NAD and 2-propanol, the kinetic parameters of the enzyme were determined to be Vmax = 29.9 Units mg-1 at 25°C and pH 8.5, KmNAD = 0.36 mm and Km2-propanol = 0.19 mm. © 1991.