Structural basis of ordered binding of donor and acceptor substrates to the retaining glycosyltransferase, α-1,3-galactosyltransferase

Bovine α-1,3-galactosyltransferase (α3GT) catalyzes the synthesis of the α-galactose (α-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 α3GT in a crystal form with limited res olution and considerable disorder suggested the possible formation of a β-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 α3GT with donor substrate, UDP-galactose, UDP-glucose, and two acceptor substrates, lactose and Nacetyllactosamine, at resolutions up to 1.46 Å. 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 β-galactose and α-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 α3GT.