NADH-dependent reduction of D-proline in Clostridium sticklandii. Reconstitution from three fractions containing NADH dehydrogenase, D-proline reductase, and a third protein factor.
The enzyme system from Clostridium sticklandii catalyzing the NADH-dependent reduction of D-proline was co-purified by chromatography on DEAE-cellulose at pH 8.2 and ammonium sulfate fractionation, and resolved into fractions containing three different protein components, NADH dehydrogenase, D-proline reductase and a third protein factor, by chromatography on DEAE-cellulose at pH 7.0. Upon recombination of the fractions containing the three different protein components, the NADH-dependent reduction of D-proline was successfully reconstituted. The NADH dehydrogenase fractions oxidized NADH in the presence of artificial electron acceptors, and were inhibited by p-hydroxymercuriphenylsulfonate (50% at 80 nM). They contained 3--4 different enzyme bands as revealed by polyacrylamide-gel electropherograms stained with the NADH-dependent reduction of 2,3,5-triphenyltetrazolium chloride. D-Proline reduction was also coupled to a leuco-methylene blue-generating system containing D-glucose and glucose-oxidase (EC 1.1.3.4). Circumstantial evidence indicated that, among the clostridial proteins, only D-proline reductase and the third protein factor were needed for this reaction.
D001426:Bacterial Proteins / Q000378:metabolism*; D002474:Cell-Free System; D003013:Clostridium / Q000378:metabolism*; D003579:Cytochrome Reductases / Q000378:metabolism*; D004579:Electron Transport; D008751:Methylene Blue / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D009245:NADH Dehydrogenase / Q000378:metabolism*; D010084:Oxidation-Reduction; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000378:metabolism*; D011392:Proline / Q000378:metabolism*; D011394:Proline Oxidase / Q000378:metabolism*; D013237:Stereoisomerism
D001426:Bacterial Proteins; D009243:NAD; D011392:Proline; D000587:Oxidoreductases Acting on CH-NH Group Donors; D011394:Proline Oxidase; D003579:Cytochrome Reductases; D009245:NADH Dehydrogenase; D008751:Methylene Blue