Publications

• Structure and catalytic cycle of beta-1,4-galactosyltransferase.

  Ramakrishnan B., Boeggeman E., Ramasamy V., Qasba P.K.

  Beta-1,4-galactosyltransferase-1, a housekeeping enzyme that functions in the synthesis of glycoconjugates, has two flexible loops, one short and one long. Upon binding a metal ion and UDP-galactose, the loops change from an open to a closed conformation, repositioning residues to lock the ligands ... >> More

  Beta-1,4-galactosyltransferase-1, a housekeeping enzyme that functions in the synthesis of glycoconjugates, has two flexible loops, one short and one long. Upon binding a metal ion and UDP-galactose, the loops change from an open to a closed conformation, repositioning residues to lock the ligands in place. Residues at the N-terminal region of the long loop form the metal-binding site and those at the C-terminal region form a helix, which becomes part of the binding site for the oligosaccharide acceptor; the remaining residues cover the bound sugar-nucleotide. After binding of the oligosaccharide acceptor and transfer of the galactose moiety, the product disaccharide unit is ejected and the enzyme returns to the open conformation, repeating the catalytic cycle. << Less

  Curr Opin Struct Biol 14:593-600(2004) [PubMed] [EuropePMC]

• Cloning of a novel member of the UDP-galactose:beta-N-acetylglucosamine beta1,4-galactosyltransferase family, beta4Gal-T4, involved in glycosphingolipid biosynthesis.

  Schwientek T., Almeida R., Levery S.B., Holmes E.H., Bennett E., Clausen H.

  A novel putative member of the human UDP-galactose:beta-N-acetylglucosamine beta1,4-galactosyltransferase family, designated beta4Gal-T4, was identified by BLAST analysis of expressed sequence tags. The sequence of beta4Gal-T4 encoded a type II membrane protein with significant sequence similarity ... >> More

  A novel putative member of the human UDP-galactose:beta-N-acetylglucosamine beta1,4-galactosyltransferase family, designated beta4Gal-T4, was identified by BLAST analysis of expressed sequence tags. The sequence of beta4Gal-T4 encoded a type II membrane protein with significant sequence similarity to other beta1,4-galactosyltransferases. Expression of the full coding sequence and a secreted form of beta4Gal-T4 in insect cells showed that the gene product had beta1,4-galactosyltransferase activity. Analysis of the substrate specificity of the secreted form revealed that the enzyme catalyzed glycosylation of glycolipids with terminal beta-GlcNAc; however, in contrast to beta4Gal-T1, -T2, and -T3, this enzyme did not transfer galactose to asialo-agalacto-fetuin, asialo-agalacto-transferrin, or ovalbumin. The catalytic activity of beta4Gal-T4 with monosaccharide acceptor substrates, N-acetylglucosamine as well as glucose, was markedly activated in the presence of alpha-lactalbumin. The genomic organization of the coding region of beta4Gal-T4 was contained in six exons. All intron/exon boundaries were similarly positioned in beta4Gal-T1, -T2, and -T3. beta4Gal-T4 represents a new member of the beta4-galactosyltransferase family. Its kinetic parameters suggest unique functions in the synthesis of neolactoseries glycosphingolipids. << Less

  J. Biol. Chem. 273:29331-29340(1998) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• The role of tryptophan 314 in the conformational changes of beta1,4-galactosyltransferase-I.

  Ramasamy V., Ramakrishnan B., Boeggeman E., Qasba P.K.

  beta1,4-Galactosyltransferase-I (beta4Gal-T1) undergoes critical conformational changes upon substrate binding from an open conformation (conf-I) to the closed conformation (conf-II). This change involves two flexible loops: the small (residues 313-316) and the long loop (residues 345-365). Upon s ... >> More

  beta1,4-Galactosyltransferase-I (beta4Gal-T1) undergoes critical conformational changes upon substrate binding from an open conformation (conf-I) to the closed conformation (conf-II). This change involves two flexible loops: the small (residues 313-316) and the long loop (residues 345-365). Upon substrate binding, Trp314 in the small flexible loop moves towards the catalytic pocket and interacts with the donor and the acceptor substrates. For a better understanding of the role played by Trp314 in the conformational changes of beta4Gal-T1, we mutated it to Ala and carried out substrate-binding, proteolytic and crystallographic studies. The W314A mutation reduces the enzymatic activity, binding to substrates and to the modifier protein, alpha-lactalbumin (LA), by over 99%. The limited proteolysis with Glu-C or Lys-C proteases shows differences in the rate of cleavage of the long loop of the wild-type and mutant W314A, indicating conformational differences in the region between the two proteins. Without substrate, the mutant crystallizes in a conformation (conf-I') (1.9A resolution crystal structure), that is not identical with, but close to an open conformation (conf-I), whereas its complex with the substrates and alpha-lactalbumin, crystallizes in a conformation (2.3A resolution crystal structure) that is identical with the closed conformation (conf-II). This study shows the crucial role Trp314 plays in the conformational state of the long loop, in the binding of substrates and in the catalytic mechanism of the enzyme. << Less

  J. Mol. Biol. 331:1065-1076(2003) [PubMed] [EuropePMC]

• Galactosyltransfer in mouse mastocytoma: purification and properties of N-acetyllactosamine synthetase.

  Helting T., Erbing B.

  Biochim Biophys Acta 293:94-104(1973) [PubMed] [EuropePMC]

• Isolation and characterization of UDP-galactose:N-acetylglucosamine 4 beta-galactosyltransferase activity induced in rat parotid glands treated with isoproterenol.

  Humphreys-Beher M.G.

  Chronic injection of the beta-adrenergic receptor agonist isoproterenol causes a 6-to 10-fold increase in the specific activity of the Golgi membrane marker enzyme UDP-galactose:D-glucose 4-beta-D-galactosyltransferase (EC 2.4.1.22) in rat parotid glands. Using a combination of size exclusion chro ... >> More

  Chronic injection of the beta-adrenergic receptor agonist isoproterenol causes a 6-to 10-fold increase in the specific activity of the Golgi membrane marker enzyme UDP-galactose:D-glucose 4-beta-D-galactosyltransferase (EC 2.4.1.22) in rat parotid glands. Using a combination of size exclusion chromatography and affinity chromatography on alpha-lactalbumin-agarose, the Triton X-100-solubilized enzyme was purified to homogeneity from treated and control animals. The enzyme purified from both sources had a single Mr = 60,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme isolated from control and isoproterenol-treated animals had identical amino acid compositions and isoelectric points (pI = 5.9). Neutral sugar content as determined by the phenol-sulfuric acid assay was 10%. UDP-galactose:N-acetylglucosamine 4 beta-galactosyltransferase activity purified by alpha-lactalbumin column chromatography was inhibited by the presence of 200 mM N-acetylglucosamine and by the inclusion of alpha-lactalbumin in assays using 10 mM N-acetylglucosamine as acceptor. Increased enzyme activity was immune precipitated from Triton X-100-solubilized membrane prepared from isoproterenol-treated animals versus control animals. This was also true of protein synthesized by in vitro translation of poly(A+) RNA prepared from parotid glands, indicating an increase in mRNA levels for the enzyme. The molecular mass of the immune-precipitated, in vitro translation product was determined to be 45,000 Da. << Less

  J Biol Chem 259:5797-5802(1984) [PubMed] [EuropePMC]

• Bovine galactosyl transferase. Substrate.managanese complexes and the role of manganese ions in the mechanism.

  Tsopanakis A.D., Herries D.G.

  Eur J Biochem 83:179-188(1978) [PubMed] [EuropePMC]