Formation of Dg-1-amino-2-propanol by a highly purified enzyme from Escherichia coli.

Specific enzymic formation of the I)g isomer" of I-amino-2-propanol, a part of the vitamin B12 molecule, has not yet been reported. KRASNA, ROSENBLUM AND SPRINSON z, using growing cultures of Streptomyces griseus, found that L-E15NJthreonine is effectively incorporated into the aminopropanol moiety of vitamin B12 and suggested that the amino alcohol might be formed by decarboxylase activity. A decarboxylase that converts L-threonine to Og-I-amino-2-propanol is not known. On the other hand, the conversion of L-threonine to aminoacetone is well documented 2-6. Furthermore, several studies 7-11 have demonstrated the presence of aminopropanol dehydrogenase (I-amino-2-propanol:DPN ÷ oxidoreductase) activity in a number of biological sources. Such studies, however, have all been carried out with cell suspensions, homogenates, or crude extracts and the enzymic activity observed prefeientially utilized the L isomer of aminopropanol. We recently obtained, in highly purified form, a dehydrogenase from Escherichia coli extracts that is specific for catalyzing a reversible oxidation-reduction reaction between aminoacetone and the D, isomer of I-amino-2-propanol; the equilibrium of this reaction is far in favor of amino ketone reduction. Aminoacetone hydrochloride was prepared by the method of TSCHUDY et al.12; the final product was recrystallized 3 times from a mixture of ethanol-ether . DLI-Amino-2-propanol (m.p., 0 I °) was a commercial product. The Dg and the Lg isomers of I-amino-2-propanol were individually prepared from L-threonine and D-threonine, respectively, by the procedure of CHALELUS 13. The products obtained were judged pure by the preparation of derivatives and the determination of optical rotation values. E. coli KI2 was grown on a nutrient broth medium; extracts were prepared by sonic disintegration of the cells. I-Amino-2-propanol dehydrogenase activity was measured by one of two methods: (I) colorimetric determination, by the method of MAUZERALL AND GRANICK 14, of the amount of aminoacetone formed, or (2) measurement of the rate of DPNH formation (I-amino-2-propanol oxidation) or of D P N H disappearance (aminoacetone reduction) at 340 m~. The latter assay could be used only with purified dehydrogenase preparations; the colorimetric assay was used in purifying the enzyme. A unit of activity is defined as the amount of protein required to form I m~mole of aminoacetone in 30 rain at 37°; specific activity refers to units of enzyme activity per mg of protein. We succeeded in purifying i-amino-2-propanol dehydrogenase activity over 25o-fold from E. coli extracts by procedures including ammonium sulfate fractionation, adsorption and elution from calcium phosphate gel, and column chromatography on DEAE-cellulose; purified enzyme fractions had specific activity values in the range of 10500o to ILOOOO. The purified dehydrogenase is optimally active between pH 8.0 and 8.6. The apparent equilibrium constant for the reaction