Mechanistic studies of the biosynthesis of 3,6-dideoxyhexoses in Yersinia pseudotuberculosis. Purification and characterization of CDP-6-deoxy-delta 3,4-glucoseen reductase based on its NADH:dichlorophenolindolphenol oxidoreductase activity.
CDP-6-deoxy-delta 3,4-glucoseen reductase, the key enzyme catalyzing the biosynthetic formation of CDP-ascarylose (CDP-3,6-dideoxy-L-arabino-hexose), was purified from Yersinia pseudotuberculosis by monitoring its NADH:dichlorophenolindolphenol oxidoreductase activity. A protocol consisting of DEAE-cellulose, phenyl-Sepharose, and Sephadex G-100 column chromatography yielded a mixture of two proteins. The low molecular weight protein contaminant was removed by limited tryptic digestion leaving a purified enzyme consisting of a single polypeptide with a molecular weight of 41,000. A weak, featureless uv spectrum above 300 nm suggested no common chromophoric cofactor contributes to enzyme activity and no protein-associated metals were detected. The stereospecificity of nicotinamide oxidation was determined to be pro-R stereospecific. Reduction of ferricyanide during NADH oxidation and confirmation of the intermediacy of O2- in the reaction flux suggested that enzyme-catalyzed H2O2 formation is not a direct two-electron reduction of molecular oxygen, but is rather the consequence of an enzymatic 2e-/1e- switch. The sugar deoxygenation reaction may therefore proceed through a radical mechanism.
Han
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Department of Chemistry, University of Minnesota, Minneapolis 55455.
Miller
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VP
Liu
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HW
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002845:Chromatography; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000032:analysis; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010088:Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D011808:Quinone Reductases / Q000302:isolation & purification / Q000378:metabolism*; D013053:Spectrophotometry; D013379:Substrate Specificity; D013481:Superoxides / Q000378:metabolism; D014357:Trypsin; D015011:Yersinia pseudotuberculosis / Q000201:enzymology*
D000596:Amino Acids; D008670:Metals; D009243:NAD; D013481:Superoxides; D009249:NADP; D010088:Oxidoreductases; C064052:CDP-6-deoxy-delta(3,4)-glucoseen reductase; D011808:Quinone Reductases; C033633:dichlorophenolindophenol reductase; D014357:Trypsin; D010100:Oxygen