Biosynthesis of UDP-N-acetyl-D-glucosaminuronic acid and UDP-N-acetyl-D-mannosaminuronic acid in Micrococcus luteus.
The occurrence and formation of UDP-N-acetyl-D-glucosaminuronic acid (UDP-GlcNAcA) and UDP-N-acetyl-D-mannosaminuronic acid (UDP-ManNAcA) were studied in Micrococcus luteus ATCC 4698. UDP-N-acetylhexosaminuronic acid separated from D-cycloserine-inhibited cells was shown to be a mixture of UDP-GlcNAcA and UDP-ManNAcA in the ratio of 87:13, whereas that obtained from untreated cells was a 96:4 mixture of these two nucleotides. Crude enzyme preparations obtained from the supernatant fraction of cells catalyzed the NAD+-dependent conversion of UDP-GlcNAc into UDP-GlcNAcA and UDP-ManNAcA. Studies on the partial separation and properties of enzymes revealed that UDP-GlcNAcA is synthesized directly from UDP-GlcNAc by the action of UDP-GlcNAc dehydrogenase and that UDP-ManNAcA is synthesized from UDP-GlcNAc through the successive actions of UDP-GlcNAc 2-epimerase and UDP-ManNAc dehydrogenase. However, enzymatic conversion of UDP-GlcNAcA to UDP-ManNAcA was not detected. Ammonium sulfate protects both dehydrogenases from inactivation during storage and incubation. Partially purified UDP-GlcNAc dehydrogenase required dithiothreitol and the particulate fraction for its full activity. The apparent Km values of UDP-GlcNAc dehydrogenase for UDP-GlcNAc and NAD+ were 0.28 and 1.43 mM, respectively. The optimum pH of this enzyme was higher than 9 in Tris-HCl buffer. p-Chloromercuribenzoate at 27 microM as well as 10 mM ethanol almost completely inhibited the UDP-GlcNAc dehydrogenase reaction.
D002237:Carbohydrate Dehydrogenases / Q000378:metabolism; D007536:Isomerism; D007700:Kinetics; D008837:Micrococcus / Q000201:enzymology / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D014539:Uridine Diphosphate Sugars / Q000096:biosynthesis*
D014539:Uridine Diphosphate Sugars; D009243:NAD; C046669:UDP-N-acetylglucosaminuronic acid; C020949:uridine diphosphate N-acetyl-D-mannosaminuronic acid; D002237:Carbohydrate Dehydrogenases; C023228:UDP-N-acetylmannosamine dehydrogenase; C046670:UDP-N-acetylglucosamine dehydrogenase