We thank the US Department of Energy for access to data collection facilities at the NSLS, and also M. Karwaski and S. Ryan for technical help. We thank Neose Pharmaceuticals for providing sialyllactose and CMP-NeuAc for the kinetic studies. The work was funded by the Howard Hughes Medical Institute, the Canadian Institute of Health Research and the Burroughs Wellcome Foundation (to N.S.), the Natural Sciences and Engineering Research Council and a Human Frontiers Science Grant (to S.W.) and by the National Research Council–GH (to W.W. and M.G.).

Gibson, R.P., Turkenburg, J.P., Charnock, S.J., Lloyd, R. & Davies, G.J. Insights into trehalose synthesis provided by the structure of the retaining glucosyltransferase OtsA. Chem. Biol. 9, 1337–1346 (2002).

Lloyd, K.O. & Furukawa, K. Biosynthesis and functions of gangliosides: recent advances. Glycoconj. J. 15, 627–636 (1998).

Image

Gebler, J.C. et al. Substrate-induced inactivation of a crippled β-glucosidase mutant: identification of the labeled amino acid and mutagenic analysis of its role. Biochemistry 34, 14547–14553 (1995).

Shibayama, K., Ohsuka, S., Tanaka, T., Arakawa, Y. & Ohta, M. Conserved structural regions involved in the catalytic mechanism of Escherichia coli K-12 WaaO (RfaI). J. Bacteriol. 180, 5313–5318 (1998).

Burmeister, W.P., Ruigrok, R.W. & Cusack, S. The 2.2 Å resolution crystal structure of influenza B neuraminidase and its complex with sialic acid. EMBO J. 11, 49–56 (1992).

Charnock, S.J. & Davies, G.J. Structure of the nucleotide-diphospho-sugar transferase, SpsA from Bacillus subtilis, in native and nucleotide-complexed forms. Biochemistry 38, 6380–6385 (1999).

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Uitdehaag, J.C.M. et al. X-ray structures along the reaction pathway of cyclodextrin glycosyltransferase elucidate catalysis in the α-amylase family. Nat. Struct. Biol. 6, 432–436 (1999).

Luo, Y., Li, S.C., Li, Y.T. & Luo, M. The 1.8 Å structures of leech intramolecular trans-sialidase complexes: evidence of its enzymatic mechanism. J. Mol. Biol. 285, 323–332 (1999).

Datta, A.K. & Paulson, J.C. The sialyltransferase “sialylmotif” participates in binding the donor substrate CMP-NeuAc. J. Biol. Chem. 270, 1497–1500 (1995).

Cowtan, K. DM: An automated procedure for phase improvement by density modification. Joint CCP4 and ESF-EACBM Newslett. Protein Crystallogr. 31, 34–38 (1994).

Institute for Biological Sciences, National Research Council, Room 3157, 100 Sussex Drive, Ottawa, K1A OR6, Ontario, Canada

Co bedeutung

Gilbert, M. et al. The genetic bases for the variation in the lipo-oligosaccharide of the mucosal pathogen, Campylobacter jejuni. Biosynthesis of sialylated ganglioside mimics in the core oligosaccharide. J. Biol. Chem. 277, 327–337 (2002).

Sialic acid terminates oligosaccharide chains on mammalian and microbial cell surfaces, playing critical roles in recognition and adherence. The enzymes that transfer the sialic acid moiety from cytidine-5′-monophospho-N-acetyl-neuraminic acid (CMP-NeuAc) to the terminal positions of these key glycoconjugates are known as sialyltransferases. Despite their important biological roles, little is understood about the mechanism or molecular structure of these membrane-associated enzymes. We report the first structure of a sialyltransferase, that of CstII from Campylobacter jejuni, a highly prevalent foodborne pathogen. Our structural, mutagenesis and kinetic data provide support for a novel mode of substrate binding and glycosyl transfer mechanism, including essential roles of a histidine (general base) and two tyrosine residues (coordination of the phosphate leaving group). This work provides a framework for understanding the activity of several sialyltransferases, from bacterial to human, and for the structure-based design of specific inhibitors.

Persson, K. et al. Crystal structure of the retaining galactosyltransferase LgtC from Neisseria meningitidis in complex with donor and acceptor sugar analogs. Nat. Struct. Biol. 8, 166–175 (2001).

Wiggins, C.A.R. & Munro, S. Activity of the yeast MNN1 α-1,3-mannosyltransferase requires a motif conserved in many other families of glycosyltransferases. Proc. Natl. Acad. Sci. USA 95, 7945–7950 (1998).

Nicholls, A., Sharp, K.A. & Honig, B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11, 281–296 (1991).

Ha, S., Walker, D., Shi, Y. & Walker, S. The 1.9 Å crystal structure of Escherichia coli MurG, a membrane-associated glycosyltransferase involved in peptidoglycan biosynthesis. Protein Sci. 9, 1045–1052 (2000).

Vyas, A.A. & Schnaar, R.L. Brain gangliosides: functional ligands for myelin stability and the control of nerve regeneration. Biochimie 83, 677–682 (2001).

CEO Übersetzung

Unligil, U.M. et al. X-ray crystal structure of rabbit N-acetylglucosaminyltransferase I: catalytic mechanism and a new protein superfamily. EMBO J. 19, 5269–5280 (2000).

Busch, C. et al. A common motif of eukaryotic glycosyltransferases is essential for the enzyme activity of large clostridial cytotoxins. J. Biol. Chem. 273, 19566–19572 (1998).

Gibbons, B.J., Roach, P.J. & Hurley, T.D. Crystal structure of the autocatalytic initiator of glycogen biosynthesis, glycogenin. J. Mol. Biol. 319, 463–477 (2002).

Crennell, S.J. et al. The structures of Salmonella typhimurium LT2 neuraminidase and its complexes with three inhibitors at high resolution. J. Mol. Biol. 259, 264–280 (1996).

co-chef

Horenstein, B.A. & Bruner, M. The N-acetyl neuraminyl oxocarbenium ion is an intermediate in the presence of anionic nucleophiles. J. Am. Chem. Soc. 120, 1357–1362 (1998).

Hagen, F.K., Hazes, B., Raffo, R., deSa, D. & Tabak, L.A. Structure-function analysis of the UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase. Essential residues lie in a predicted active site cleft resembling a lactose repressor fold. J. Biol. Chem. 274, 6797–6803 (1999).

Chiu, C., Watts, A., Lairson, L. et al. Structural analysis of the sialyltransferase CstII from Campylobacter jejuni in complex with a substrate analog. Nat Struct Mol Biol 11, 163–170 (2004). https://doi.org/10.1038/nsmb720

Crennell, S., Garman, E., Laver, G., Vimr, E. & Taylor, G. Crystal structure of Vibrio cholerae neuraminidase reveals dual lectin-like domains in addition to the catalytic domain. Structure 2, 535–544 (1994).

Nature Structural & Molecular Biology (Nat Struct Mol Biol) ISSN 1545-9985 (online) ISSN 1545-9993 (print)

Department of Biochemistry and Molecular Biology, University of British Columbia, 2146 Health Sciences Mall, Vancouver, V6T 1Z3, British Columbia, Canada

Coutinho, P.M., Deleury, E., Davies, G.J. & Henrissat, B. An evolving hierarchical family classification for glycosyltransferases. J. Mol. Biol. 328, 307–317 (2003).

Gosselin, S., Alhussaini, M., Streiff, M.B., Takabayashi, K. & Palcic, M.M. A continuous spectrophotometric assay for glycosyltransferases. Anal. Biochem. 220, 92–97 (1994).

Und Co Englisch

Gaskell, A., Crennell, S. & Taylor, G. The three domains of a bacterial sialidase: A β-propeller, an immunoglobulin module and a galactose-binding jelly-roll. Structure 3, 1197–1205 (1995).

Pedersen, L.C. et al. Heparan/chondroitin sulfate biosynthesis. Structure and mechanism of human glucuronyltransferase I. J. Biol. Chem. 275, 34580–34585 (2000).

EnglischDeutsch

Vrielink, A., Ruger, W., Driessen, H.P. & Freemont, P.S. Crystal structure of the DNA modifying enzyme β-glucosyltransferase in the presence and absence of the substrate uridine diphosphoglucose. EMBO J. 13, 3413–3422 (1994).

Luo, Y., Li, S.C., Chou, M.Y., Li, Y.T. & Luo, M. The crystal structure of an intramolecular trans-sialidase with a NeuAc[α]2→3Gal specificity. Structure 6, 521–530 (1998).

Gilbert, M. et al. Biosynthesis of ganglioside mimics in Campylobacter jejuni OH4384. Identification of the glycosyltransferase genes, enzymatic synthesis of model compounds, and characterization of nanomole amounts by 600-MHz 1H and 13C NMR analysis. J. Biol. Chem. 275, 3896–3906 (2000).

CodDeutsch

Endtz, H.P. et al. Molecular characterization of Campylobacter jejuni from patients with Guillain-Barre and Miller Fisher syndromes. J. Clin. Microbiol. 38, 2297–2301 (2000).

McRee, D.E. XtalView/Xfit—a versatile program for manipulating atomic coordinates and electron density. J. Struct. Biol. 125, 156–165 (1999).

Kraulis, P. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946–950 (1991).

Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

Amaya, M.F., Buschiazzo, A., Nguyen, T. & Alzari, P.M. The high resolution structures of free and inhibitor-bound Trypanosoma rangeli sialidase and its comparison with T. cruzi trans-sialidase. J. Mol. Biol. 325, 773–784 (2003).

Penner, J.L. & Aspinall, G.O. Diversity of lipopolysaccharide structures in Campylobacter jejuni. J. Infect. Dis. 176 Suppl 2, S135–S138 (1997).

Burkart, M.D. et al. Chemo-enzymatic synthesis of fluorinated sugar nucleotide: useful mechanistic probes for glycosyltransferases. Bioorg. Med. Chem. 8, 1937–1946 (2000).

Laskowski, R.A., MacArthur, M.W., Moss, D.S. & Thornton, J.M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291 (1993).

Guerry, P. et al. Phase variation of Campylobacter jejuni 81–176 lipooligosaccharide affects ganglioside mimicry and invasiveness in vitro. Infect. Immun. 70, 787–793 (2002).

Image

Watts, A.G. & Withers, S.G. The synthesis of some mechanistic probes for sialic acid processing enzymes and the labeling of a sialidase from Trypanosoma rangeli. Can. J. Chem. (in the press).

Image

Crennell, S., Takimoto, T., Portner, A. & Taylor, G. Crystal structure of the multifunctional paramyxovirus hemagglutinin-neuraminidase. Nat. Struct. Biol. 7, 1068–1074 (2000).

Brunger, A.T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. A 54, 905–921 (1998).

Timm, D.E., Liu, J., Baker, L.J. & Harris, R.A. Crystal structure of thiamin pyrophosphokinase. J. Mol. Biol. 310, 195–204 (2001).

CoDeutschPolnisch

Crennell, S.J., Garman, E.F., Laver, W.G., Vimr, E.R. & Taylor, G.L. Crystal structure of a bacterial sialidase (from Salmonella typhimurium LT2) shows the same fold as an influenza virus neuraminidase. Proc. Natl. Acad. Sci. USA 90, 9852–9856 (1993).

Horenstein, B.A. Quantum mechanical analysis of an α-carboxylate-substituted oxocarbenium ion. Isotope effects for formation of the sialyl cation and the origin of an unusually large 14C secondary isotope effect. J. Am. Chem. Soc. 119, 1101–1107 (1997).

Buschiazzo, A. et al. Structural basis of sialyltransferase activity in trypanosomal sialidases. EMBO. J. 19, 16–24 (2000).