Publications

• Characterization of a Bacillus subtilis thermosensitive teichoic acid-deficient mutant: gene mnaA (yvyH) encodes the UDP-N-acetylglucosamine 2-epimerase.

  Soldo B., Lazarevic V., Pooley H.M., Karamata D.

  The Bacillus subtilis thermosensitive mutant ts-21 bears two C-G-->T-A transitions in the mnaA gene. At the nonpermissive temperature it is characterized by coccoid cell morphology and reduced cell wall phosphate content. MnaA converts UDP-N-acetylglucosamine into UDP-N-acetylmannosamine, a precur ... >> More

  The Bacillus subtilis thermosensitive mutant ts-21 bears two C-G-->T-A transitions in the mnaA gene. At the nonpermissive temperature it is characterized by coccoid cell morphology and reduced cell wall phosphate content. MnaA converts UDP-N-acetylglucosamine into UDP-N-acetylmannosamine, a precursor of the teichoic acid linkage unit. << Less

  J. Bacteriol. 184:4316-4320(2002) [PubMed] [EuropePMC]

• Active site mutants of the 'non-hydrolyzing' UDP-N-acetylglucosamine 2-epimerase from Escherichia coli.

  Samuel J., Tanner M.E.

  The "non-hydrolyzing" bacterial UDP-N-acetylglucosamine 2-epimerase catalyzes the reversible interconversion of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylmannosamine (UDP-ManNAc). This homodimeric enzyme is allosterically activated by its substrate, UDP-GlcNAc, and it is thought that on ... >> More

  The "non-hydrolyzing" bacterial UDP-N-acetylglucosamine 2-epimerase catalyzes the reversible interconversion of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylmannosamine (UDP-ManNAc). This homodimeric enzyme is allosterically activated by its substrate, UDP-GlcNAc, and it is thought that one subunit plays a regulatory role, while that of the other plays a catalytic role. In this work, five active site mutants were prepared (D95N, E117Q, E131Q, K15A, and H213N) and analyzed in terms of their effects on binding, catalysis, and allosteric regulation. His213 appears to play a role in UDP binding and may also assist in catalysis and/or regulation, but is not a key catalytic residue. Lys15 appears to be quite important for binding. All three of the carboxylate mutants showed dramatic decreases in the value of k(cat) but relatively unaffected values of K(M). Thus, these residues are playing key roles in catalysis and/or regulation. In the case of E117Q, the reaction intermediates are released into solution at a rate comparable to that of the overall catalysis. This may indicate that Glu117 plays the role as an acid/base catalyst in the second step of the UDP-GlcNAc epimerization reaction. All three carboxylate mutants were found to exhibit impaired allosteric control. << Less

  Biochim. Biophys. Acta 1700:85-91(2004) [PubMed] [EuropePMC]

• Biosynthesis of enterobacterial common antigen in Escherichia coli. Biochemical characterization of Tn10 insertion mutants defective in enterobacterial common antigen synthesis.

  Meier-Dieter U., Starman R., Barr K., Mayer H., Rick P.D.

  Twelve independent Tn10 insertion mutants of Escherichia coli K12 were isolated that were defective in the synthesis of enterobacterial common antigen (ECA). The mutants were identified by screening a random pool of Tn10 insertion mutants for their ECA phenotype using a colony-immunoblot assay. Al ... >> More

  Twelve independent Tn10 insertion mutants of Escherichia coli K12 were isolated that were defective in the synthesis of enterobacterial common antigen (ECA). The mutants were identified by screening a random pool of Tn10 insertion mutants for their ECA phenotype using a colony-immunoblot assay. All 12 of the Tn10 insertion mutants were found to be located in the chromosomal region of the rff-rfe genes. Four of the Tn10 insertions were in rfe genes while the remaining eight Tn10 insertions were in rff genes. All of the rfe::Tn10 insertion mutants were defective in the synthesis of GlcNAc-pyrophosphorylundecaprenol (C55-PP-GlcNAc, lipid I), the first lipid-linked intermediate involved in ECA synthesis. Biochemical characterization of the rff::Tn10 insertion mutants revealed that they were defective in various steps of ECA synthesis subsequent to the synthesis of lipid I. These defects included: (i) the inability to synthesize UDP-ManNAcA due to Tn10 insertions in the structural genes for UDP-GlcNAc-2-epimerase (rffE) and UDP-ManNAcA (N-acetyl-D-mannosaminuronic acid) dehydrogenase (rffD), (ii) defects in the synthesis of C55-GlcNAc-ManNAcA (lipid II) due to insertion of transposon Tn10 in the structural gene for the UDP-ManNAcA transferase (rffM), (iii) the inability to synthesize TDP-Fuc4NAc (4-acetamido-4,6-dideoxy-D-galactose) due to Tn10 insertions in the structural gene for the transaminase that catalyzes the conversion of TDP-4-keto-6-deoxy-D-glucose to TDP-4-amino-4,6-dideoxy-D-galactose (rffA), and (iv) defects in steps subsequent to the synthesis of C55-GlcNAc-ManNAcA-Fuc4NAc (lipid III). In addition, a re-examination of a mutant possessing the rff-726 lesion revealed that it was defective in the synthesis of lipid III due to a defect in the structural gene for the Fuc4NAc transferase (rffT). << Less

  J. Biol. Chem. 265:13490-13497(1990) [PubMed] [EuropePMC]

• The structure of UDP-N-acetylglucosamine 2-epimerase reveals homology to phosphoglycosyl transferases.

  Campbell R.E., Mosimann S.C., Tanner M.E., Strynadka N.C.

  Bacterial UDP-N-acetylglucosamine 2-epimerase catalyzes the reversible epimerization at C-2 of UDP-N-acetylglucosamine (UDP-GlcNAc) and thereby provides bacteria with UDP-N-acetylmannosamine (UDP-ManNAc), the activated donor of ManNAc residues. ManNAc is critical for several processes in bacteria, ... >> More

  Bacterial UDP-N-acetylglucosamine 2-epimerase catalyzes the reversible epimerization at C-2 of UDP-N-acetylglucosamine (UDP-GlcNAc) and thereby provides bacteria with UDP-N-acetylmannosamine (UDP-ManNAc), the activated donor of ManNAc residues. ManNAc is critical for several processes in bacteria, including formation of the antiphagocytic capsular polysaccharide of pathogens such as Streptococcus pneumoniae types 19F and 19A. We have determined the X-ray structure (2.5 A) of UDP-GlcNAc 2-epimerase with bound UDP and identified a previously unsuspected structural homology with the enzymes glycogen phosphorylase and T4 phage beta-glucosyltransferase. The relationship to these phosphoglycosyl transferases is very intriguing in terms of possible similarities in the catalytic mechanisms. Specifically, this observation is consistent with the proposal that the UDP-GlcNAc 2-epimerase-catalyzed elimination and re-addition of UDP to the glycal intermediate may proceed through a transition state with significant oxocarbenium ion-like character. The homodimeric epimerase is composed of two similar alpha/beta/alpha sandwich domains with the active site located in the deep cleft at the domain interface. Comparison of the multiple copies in the asymmetric unit has revealed that the epimerase can undergo a 10 degrees interdomain rotation that is implicated in the regulatory mechanism. A structure-based sequence alignment has identified several basic residues in the active site that may be involved in the proton transfer at C-2 or stabilization of the proposed oxocarbenium ion-like transition state. This insight into the structure of the bacterial epimerase is applicable to the homologous N-terminal domain of the bifunctional mammalian UDP-GlcNAc "hydrolyzing" 2-epimerase/ManNAc kinase that catalyzes the rate-determining step in the sialic acid biosynthetic pathway. << Less

  Biochemistry 39:14993-15001(2000) [PubMed] [EuropePMC]

• Enzymatic formation of uridine diphosphate N-acetyl-D-mannosamine.

  Kawamura T., Kimura M., Yamamori S., Ito E.

  An enzyme that catalyzes the interconversion of UDP-N-acetyl-D-glucosamine and UDP-N-acetyl-D-mannosamine was purified about 700-fold from the supernatant fraction of Bacillus cereus, and the properties of this enzyme were studied. This enzyme was not stimulated by NAD+, NADH, or any metal ions. T ... >> More

  An enzyme that catalyzes the interconversion of UDP-N-acetyl-D-glucosamine and UDP-N-acetyl-D-mannosamine was purified about 700-fold from the supernatant fraction of Bacillus cereus, and the properties of this enzyme were studied. This enzyme was not stimulated by NAD+, NADH, or any metal ions. The optimum pH was between 7.5 and 8.0. At equilibrium of the reaction, the ratio of UDP-N-acetylglucosamine to UDP-N-acetylmannosmaine was about 9:1. The enzyme was inactive toward free N-acetylhexosamines, their phosphate esters, UDP-glucose, and UDP-N-acetylgalactosamine. A stimulatory role of UDP-N-acetylglucosamine was demonstrated. In the reaction with UDP-N-acetylglucosamine, the rate as a function of substrate concentration showed a sigmoidal relationship with a Hill coefficient of 1.8 and an apparent Km value for UDP-N-acetylglucosamine of 1.1 mM. The reverse reaction with UDP-N-acetylmannosamine required the presence of UDP-N-acetylglucosamine. The UDP-N-acetylglucosamine concentration required for half-maximal activation was about 0.5 mM. The apparent Km for UDP-N-acetylmannosamine measured in the presence of 0.5 mM UDP-N-acetylglucosamine was 0.22mM. Other nucleotides or hexosamine derivatives were not stimulatory. The same activity was found in cell extracts from several bacterial species. << Less

  J Biol Chem 253:3595-3601(1978) [PubMed] [EuropePMC]