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

beta 1,4-galactosyltransferase T1 (beta 4Gal-T1, EC 2.4.1.90/38), a Golgi r esident membrane-bound enzyme, transfers galactose from uridine diphosphoga lactose to the terminal beta-N-acetylglucosamine residues forming the poly- N-acetylactosamine core structures present in glycoproteins and glycosphing olipids. In mammals, beta 4Gal-T1 binds to alpha-lactalbumin, a protein tha t is structurally homologous to lyzozyme, to produce lactose, beta Gal-T1 i s a member of a large family of homologous beta 4galactosyltransferases tha t use different types of glycoproteins and glycolipids as substrates. Here we solved and refined the crystal structures of recombinant bovine beta 4Ga l-T1 to 2.4 Angstrom resolution in the presence and absence of the substrat e uridine diphosphogalactose. The crystal structure of the bovine substrate -free beta 4Gal-T1 catalytic domain showed a new fold consisting of a singl e conical domain with a large open pocket at its base. In the substrate-bou nd complex, the pocket encompassed residues interacting with uridine diphos phogalactose. The structure of the complex contained clear regions of elect ron density for the uridine diphosphate portion of the substrate, where its beta-phosphate group was stabilized by hydrogen-bonding contacts with cons erved residues including the Asp252ValAsp254 moth, These results help the i nterpretation of engineered beta 4Gal-T1 point mutations, They suggest a me chanism possibly involved in galactose transfer and enable identification o f the critical amino acids involved in alpha-lactalbumin interactions.