Preliminary joint neutron time-of-flight and X-ray crystallographic study of human ABO(H) blood group A glycosyltransferase

Abstract

The biosyntheses of oligosaccharides and glycoconjugates are conducted by glycosyltransferases. These extraordinarily diverse and widespread enzymes catalyze the formation of glycosidic bonds through the transfer of a monosaccharide from a donor molecule to an acceptor molecule, with the stereochemistry about the anomeric carbon being either inverted or retained. Human ABO(H) blood group A α‐1,3‐N‐acetylgalactosaminyltransferase (GTA) generates the corresponding antigen by the transfer of N‐acetylgalactosamine 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 initiated at the Protein Crystallography Station (PCS) at Los Alamos Neutron Science Center (LANSCE). A large single crystal was subjected to H/D exchange prior to data collection and time‐of‐flight neutron diffraction data were collected to 2.5 Å resolution at the PCS to ∼85% overall completeness, with complementary X‐ray diffraction data collected from a crystal from the same drop and extending to 1.85 Å resolution. Here, the first successful neutron data collection from a glycosyltransferase is reported.

Geometric Attributes of Retaining Glycosyltransferase Enzymes Favor an Orthogonal Mechanism

“…With the exception of the GTA neutron diffraction studies [44] hydrogen atoms are not directly observed, so distances are given between centers of non-hydrogen atoms. Italic enzyme names indicate the model did not contain an acceptor molecule.…”

“…A neutron structure of the human retaining enzyme GTA at LANCE PCS (PDB 4DHH associated with [44]) has been reported. More detailed analysis of this structure has revealed an aprotic active site that appears to be incompatible with a dissociative mechanism.…”