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.