Abstract
Bovine alpha-1,3-galactosyltransferase (alpha3GT) catalyzes the synthesis of the alpha-galactose (alpha-Gal) epitope, the target of natural human antibodies. It represents a family of enzymes, including the histo blood group A and B transferases, that catalyze retaining glycosyltransfer reactions of unknown mechanism. An initial study of alpha3GT in a crystal form with limited resolution and considerable disorder suggested the possible formation of a beta-galactosyl-enzyme covalent intermediate (Gastinel, L. N., Bignon, C., Misra, A. K., Hindsgaul, O., Shaper, J. H., and Joziasse, D. H. (2001) EMBO J. 20, 638-649). Highly ordered structures are described for complexes of alpha3GT with donor substrate, UDP-galactose, UDP- glucose, and two acceptor substrates, lactose and N-acetyllactosamine, at resolutions up to 1.46 A. Structural and calorimetric binding studies suggest an obligatory ordered binding of donor and acceptor substrates, linked to a donor substrate-induced conformational change, and the direct participation of UDP in acceptor binding. The monosaccharide-UDP bond is cleaved in the structures containing UDP-galactose and UDP-glucose, producing non-covalent complexes containing buried beta-galactose and alpha-glucose. The location of these monosaccharides and molecular modeling suggest that binding of a distorted conformation of UDP-galactose may be important in the catalytic mechanism of alpha3GT.
Highlights
Bovine ␣-1,3-galactosyltransferase (␣3GT) catalyzes the synthesis of the ␣-galactose (␣-Gal) epitope, the target of natural human antibodies
Ordered structures are described for complexes of ␣3GT with donor substrate, UDP-galactose, UDPglucose, and two acceptor substrates, lactose and Nacetyllactosamine, at resolutions up to 1.46 Å
Structures of ␣3GT Complexes with Donor Substrate, UDPGal, and UDP-Glc—The UDP-Gal1⁄7enzyme complex was prepared by co-crystallization, and the UDP-Glc complex was prepared by soaking crystals initially grown in the presence of UDP with UDP-Glc, yet the structures are closely similar to each other and to the ␣3GT1⁄7Mn2ϩ1⁄7UDP complex
Summary
X-ray Crystallography—The catalytic domain of bovine ␣3GT (residues 80 –368) was expressed in Escherichia coli and purified as previously described [20] and stored at Ϫ20 °C in 20 mM MES-NaOH buffer (pH 6) in 50% glycerol. l of the protein at 5 mg/ml in 20 mM MES-NaOH buffer, pH 6.0, 10% glycerol, containing 10 mM UDP and 0.1 mM MnCl2, with an equal volume of a reservoir solution containing 5% PEG 6000 and 0.1 M Tris-HCl, pH 8.0. The data to parameter ratio, Ͼ2, enabled us to carry out anisotropic refinement on ADPs that reduced both R-factors by at least 4.5% thereby justifying the use of this method of refinement. The binding isotherm was generated by plotting the corrected heats of binding against the ratio of ligand to enzyme. Software supplied by the manufacturer (Origin version 5.0 from Microcal Inc.) was used to calculate dissociation constants (Kd), enthalpies of binding (⌬H), stoichiometry (N), and entropy of binding (⌬S)
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