Purification and properties of l-1-aminopropan-2-ol. NAD oxidoreductase from a pseudomonad grown on DL-1-aminopropan-2-ol.

Abstract

SUMMARY: Two pseudomonad strains grown on DL- I -aminopropan-2-01 as sole source of carbon + nitrogen provided rich sources of L (+)- I -aminopropan- 2-01 : NAD+ oxidoreductase. The activity of this enzyme in crude extracts of Pseudomona's sp. strain NCIB 8858 was about 400 mpmoles 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-o1 and NAD+ were about 0· 1 and 0·3 mM, respectively. Activity with NADP+ as the coenzyme was about the same as that with NADf, the Km being about 0·2 mM.The e nzyme was inactive with a-NAD+. Activity was unaffected by a variety of thiols, thiol-alkylating and metalchelating 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 m M , 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.