Biosynthesis of UDP-N-Acetyl-D-Glucosaminuronic Acid and UDP-N-Acetyl-D-Mannosaminuronic Acid in Micrococcus luteus1

Abstract

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.

Biochemical Characterization of WbpA, a UDP-N-acetyl-d-glucosamine 6-Dehydrogenase Involved in O-antigen Biosynthesis in Pseudomonas aeruginosa PAO1

“…WbpA has a broad pH optimum of 8 -9, which correlates well with the reported pH optima of UDP-D-GlcNAc 6-dehydrogenase from A. georgiopolitanum (pH 9) and from M. luteus (pH 8.8), and of UDP-glucose dehydrogenase from E. coli (pH 9) (28,29,33). This also correlates well with the results of WbpO (pH 8.5), another dehydrogenase that was previously characterized by our group (31).…”

“…Closer examination revealed that WbpA requires monovalent cations, either NH 4 ϩ or K ϩ in particular, for activity. This observation is in contrast to previous suggestions that ammonium sulfate was required to prevent loss of UDP-D-GlcNAc 6-dehydrogenase activity in extracts from M. luteus (29). Furthermore, our results are consistent with those of other sugar-nucleotide modifying enzymes including MurB, a UDP-N-acetylenoylpyruvylglucosamine reductase from E. coli and Streptococcus pneumoniae that requires K ϩ , NH 4 ϩ , or Rb ϩ (36,37).…”

“…WbpA has a K 0.5 of 94 M for its specific substrate, UDP-DGlcNAc, which is lower than those observed for the M. luteus (280 M) or A. geogiopolitanum (500 M) enzymes (28,29) indicating a higher binding efficiency between WbpA and its substrate. This result can be expected since the latter two enzymes were only partially purified when analyzed.…”

“…Although UDP-D-GlcNAc 6-dehydrogenase activity was previously observed in both Achromobacter georgiopolitanum and Micrococcus luteus (28,29), the enzyme itself was never identified. Therefore, this is the first report of identification, purification, and in-depth biochemical characterization of this enzyme, including detailed analysis of cofactor requirements.…”

Formation of UDP-2-acetamido-2-deoxy-L-galactose and UDP-2-acetamido-2-deoxy-L-galacturonic acid by Pseudomonas aeruginosa

“…A somewhat similar situation exists in Micrococcus luteus, in which UDP-GlcNAc is converted via UDPManNAc to UDP-ManNAcA; the last compound serves as a donor in teichoic acid synthesis. In a separate reaction, M. luteus oxidizes UDP-GlcNAc to UDP-GlcNAcA; however, since M. luteus does not contain GlcNAcA residues, no function has yet been assigned to UDP-GlcNAcA (9).…”