Neutron Structure of Retaining Glycosyltransferase GTA

With at most one electron, hydrogen atoms can be difficult to observe in protein structures by X-ray crystallography even at atomic resolution. This can pose a barrier to the critical evaluation of enzymatic mechanisms that involve proton translocation as the ionization states of active site residues cannot always be reliably determined by the chemical environment in which they reside.

One such class of enzyme is the glycosyltransferase. Glycosyltransferases catalyze the biosynthesis of oligosaccharides and glycoconjugates by the transfer of a monosaccharide unit from an activated donor molecule to an acceptor molecule with high stereospecificity. The anomeric stereochemistry of the donor sugar will either be inverted or retained upon formation of the new glycosidic linkage, depending on the enzyme. Although a consensus has been reached on an S_N2 mechanism used by inverting glycosyltransferases, the nature of the retaining mechanism is still a matter of debate.

The model retaining enzyme human ABO(H) blood group A α-1,3-N-acetylgalactosaminyl- transferase (GTA) generates the blood group A antigen by the transfer of N-acetyl-galactosamine from UDP-GalNAc to the blood group H antigen. To understand better how specific active-site-residue protons and hydrogen-bonding patterns affect substrate recognition and catalysis, neutron diffraction studies were conducted at the Protein Crystallography Station (PCS) at Los Alamos Neutron Science Center (LANSCE).

This is the first study of a retaining glycosyltransferase using combined X-ray and neutron diffraction data. These data provide the first unambiguous assignment of protons and the causative hydrogen-bond patterns in a glycosyltransferase active site.