Crystal structures of the bovine beta 4galactosyltransferase catalytic domain and its complex with uridine diphosphogalactose

Abstract

β1,4‐galactosyltransferase T1 (β4Gal‐T1, EC 2.4.1.90/38), a Golgi resident membrane‐bound enzyme, transfers galactose from uridine diphosphogalactose to the terminal β‐N‐acetylglucosamine residues forming the poly‐N‐acetyllactosamine core structures present in glycoproteins and glycosphingolipids. In mammals, β4Gal‐T1 binds to α‐lactalbumin, a protein that is structurally homologous to lyzozyme, to produce lactose. β4Gal‐T1 is a member of a large family of homologous β4galactosyltransferases that use different types of glycoproteins and glycolipids as substrates. Here we solved and refined the crystal structures of recombinant bovine β4Gal‐T1 to 2.4 Å resolution in the presence and absence of the substrate uridine diphosphogalactose. The crystal structure of the bovine substrate‐free β4Gal‐T1 catalytic domain showed a new fold consisting of a single conical domain with a large open pocket at its base. In the substrate‐bound complex, the pocket encompassed residues interacting with uridine diphosphogalactose. The structure of the complex contained clear regions of electron density for the uridine diphosphate portion of the substrate, where its β‐phosphate group was stabilized by hydrogen‐bonding contacts with conserved residues including the Asp252ValAsp254 motif. These results help the interpretation of engineered β4Gal‐T1 point mutations. They suggest a mechanism possibly involved in galactose transfer and enable identification of the critical amino acids involved in α‐lactalbumin interactions.