Structural basis of ordered binding of donor and acceptor substrates to the retaining glycosyltransferase, alpha-1,3-galactosyltransferase.
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.
Boix
Ester
E
Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom.
Zhang
Yingnan
Y
Swaminathan
G Jawahar
GJ
Brew
Keith
K
Acharya
K Ravi
KR
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001665:Binding Sites; D002417:Cattle; D005700:Galactosyltransferases / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D013379:Substrate Specificity; D013499:Surface Properties
D000906:Antibodies; D005700:Galactosyltransferases; C038887:N-acetyllactosamine alpha-D-galactosyltransferase