Purification and Properties of L-1-Aminopropan-2-o1:NAD Oxidoreductase from a Pseudomonad Grown on DL-1-Aminopropan-2-o1

SUMMARY: Two pseudomonad strains grown on DL-I-aminopropan-2-o1 as sole source of carbon + nitrogen provided rich sources of L(+)-I-aminopropan-2-o1: NAD+ oxidoreductase. The activity of this enzyme in crude extracts of Pseudomonas sp. strain NCIB 8858 was about 400 mμmoles aminoacetone formed/mg. protein/min., under optimum conditions. Growth on media containing other carbon sources commonly repressed enzyme formation. Enzyme formation was induced by incubating succinate-grown bacteria in media containing DL-I-aminopropan-2-ol, aminoacetone or L-threonine; but induction was inhibited by DL-2-hydroxy-2-phenylethylamine or DL-2-phenylserine. The enzyme was purified twenty-fold by treatment with protamine sulphate and ammonium sulphate followed by gel-filtration. The molecular weight of the enzyme was estimated to be 70 to 80 thousand. The partly purified enzyme was optimally active at pH 9.5. The Km values for DL-I-aminopropan-2-ol and NAD+ were about 0.1 and 0.3 mM, respectively. Activity with NADP+ as the coenzyme was about the same as that with NAD+, the Km being about 0.2 mM. The enzyme was inactive with α-NAD+. Activity was unaffected by a variety of thiols, thiol-alkylating and metal-chelating agents. The enzyme was virtually inactive with twenty-one potential substrates but oxidized DL-I-aminobutan-2-ol, DL-2-hydroxy-2-phenylethylamine and DL-phenylserine at significant rates and DL-I,3-diaminopropan-2-ol and DL-3-hydroxybutyrate slowly. Enzyme activity towards I-amino-propan-2-ol was stereospecific for the L(+)-isomer, which was oxidized about six times more rapidly than the racemic substrate. The D(-)-enantiomorph was a competitive inhibitor of the reaction, Ki = about 0.3 mM, and no dehydrogenase activity towards this isomer was observed with either purified enzyme preparations or crude cell-free extracts. Aminoacetone-dependent oxidation of NADH occurred optimally at pH 5 in acetate buffer; a second peak of activity was found at pH 8.4 in diethanolamine + HCl buffers. Several buffers appeared to inhibit activity at pH 7.5. 2′-Aminoacetophenone was active as a substrate, but 5-aminolaevulate had little activity. NADPH was also active as coenzyme.