1887

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 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.

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/content/journal/micro/10.1099/00221287-54-1-115
1968-11-01
2022-01-22
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References

  1. ANDREWS P. 1965; The gel filtration behaviour of proteins related their molecular weights over a wide range.. Biochem. J. 96:595
    [Google Scholar]
  2. CHANCE B., MAEHLY A. C. 1955; Assay of catalases and peroxidases.. Meth. Enzymol. 2:764
    [Google Scholar]
  3. ELLIOTT W. H. 1958; A new threonine metabolite.. Biochem. biophys. Acta 29, 44:6
    [Google Scholar]
  4. HIGGINS I. J., PICKARD M. A., TURNER J. M. 1966; Aminoacetone formation and utilization by pseudomonads grown on DL-1-aminopropan-2-ol.. J. gen. Microbiol. 54:105
    [Google Scholar]
  5. HIGGINS I. J., PICKARD M. A., TURNER J. M., WILLETTS A. J. 1966; 1-Aminopropan-2-ol; NAD oxidoreductase.. Biochem. J. 9927
    [Google Scholar]
  6. HORECKER B. J., KORNBERG A. 1948; The extinction coefficients of the reduced band of pyridine nucleotides.. J. biol. Chem. 175:385
    [Google Scholar]
  7. LINEWEAVER H., BURK D. 1934; The determination of enzyme dissociation constants.. J. Am. chem. Soc. 56:658
    [Google Scholar]
  8. PICKARD M. A., HIGGINS I. J., TURNER J. M. 1966; Microbial metabolism of 1-aminopropan- 2-0I. J.. gen. Microbiol, 45:i
    [Google Scholar]
  9. RACKER E. 1955; Alcohol dehydrogenase from baker's yeast.. Meth. Enzymol. 1:500
    [Google Scholar]
  10. SULLIVAN R. H. 1963; Resolution of racemic aminoisopropanol.. U.S. Patent 3, 116, 332, Cl, 260–584
    [Google Scholar]
  11. TURNER J. M. 1966a; A. minoacetone production by micro-organisms.. Biochem. J 98:7P
    [Google Scholar]
  12. TURNER J. M. 1966b; Microbial metabolism of amino ketones: aminoacetone formation from 1-aminopropan-2-01 by a dehydrogenase in Escherichia coli. . Biochem. J. 99:427
    [Google Scholar]
  13. TURNER J. M. 1967; Microbial metabolism of amino ketones: L-I-aminopropan-2-01 and L-threoninedehydrogenase in Escherichia coli.. Biochem. J. 104:112
    [Google Scholar]
  14. TURNER J. M., WILLETTS A. J. 1967; Amino ketone formation and aminopropanol-dehydrogenase activity in rat liver preparations.. Biochem. J. 102:511
    [Google Scholar]
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