During the FY 1998-1999 we continued the investigations on the structural and functional aspects of the members of beta-1,4-galactosyltransferase enzyme (beta4Gal-T) family, a sub-family of glycosyltransferase super-family that is involved in the synthesis of complex oligosaccharides of glycoconjugates. Each beta4Gal-T family member transfers galactose from UDP-alpha-D-galactose to N acetylglucosamine (GlcNAc), with an ?inversion? of the anomeric configuration at the C1 carbon atom of galactose, generating a beta1-4-linkage. However, each member shows differences in the sugar acceptor specificities and interacts differently with a-lactalbumin (alpha-LA), which modifies the acceptor substrate specificity of the enzyme to glucose. In addition of its transfer of Gal from UDP-alpha-D-galactose to GlcNAc, beta4Gal-T1 also transfers Glc from UDP-alpha-D-glucose to GlcNAc comprising its glucosyltransferase (Glc-T) activity, albeit at an efficiency of 0.3-0.4% of Gal-T activity. We have shown that in the presence of alpha-LA the Glc-T activity of beta4Gal-T1 is enhanced by nearly 30 fold, corresponding to an efficiency of about 10% of the Gal-T activity of the enzyme. By site directed mutagenesis we have identified Trp198 in the recombinant beta4Gal-T1, located within a non-conserved aromatic region, 197YWLY200, to be at least partially involved in binding both the sugar moiety of the sugar-nucleotide donor and the alpha-LA. The apparent Km of W198A mutant with respect to both UDP-alpha Gal and UDP-alpha-Glc in the GalT- and GlcT-reactions, respectively, decreases compared to the wild type protein. The catalytic turnover number, Kcat, and the catalytic efficiency, Kcat/Km, both decrease significantly with the mutant protein. However, in the presence of alpha-LA, the apparent Km for UDP-alpha-Gal and Glc increases, and as well the apparent Km for alpha-LA increases compared to the wild type, indicating that Trp-198 may be positioned at the interface of the sugar-nucleotide binding site and the site at which beta4Gal-T1 interacts with alpha-LA forcing a conformational change(s) within the enzyme-metal-sugar nucleotide complex in a way that dictates the selection of the acceptor molecule for the reaction. We have also shown that mutation of Cys-342 to Thr increases the in vitro folding efficiency of beta4Gal-T1 from the inclusion bodies by 2 to 3 fold while maintaining the structural integrity and enzymatic activity of the protein. The enzymatic activity has an absolute requirement for Mn+2. Other metal ions, e.g. Co+2, Zn+2, Cd+2, and Fe+2, also activate beta4Gal-T1, albeit to a lesser extent compared to Mn+2. Two metal binding sites, I and II, have been proposed for the beta4-Gal-T1. Site I has an absolute requirement for manganese (Kd = 2 x 10-6 M) and does not bind Ca+2. The second metal binding site can bind Ca+2 and activate the enzyme at low Mn+2 concentrations (10-5 M). Kinetic studies show that the Gal-T activity of the wild type and the mutants of site I, the DXD motif, D244N and D252E, can be activated by Ca+2 in the presence of a low concentration of Mn+2 (2 microM). However, the mutants of site II, E317D, D320N and D320E cannot be activated by Ca+2, even at higher Mn+2 concentrations (20 microM). On the other hand at a fixed Mn+2 concentration (20 microM), Co+2 activates D320N, and D320E to the same level as in the absence of Mn+2, but the wild type GT-d129 is inhibited. In recent years, taking advantage of EST sequences, at least six different family members of beta-4Gal-T, T1 to T6, have been identified in the human genome which exhibit high sequence identity in the catalytic domain of enzyme (80% to 40%). Each member of the family has been shown to be expressed in a tissue specific manner. Among the family members beta4Gal-T4 has only 8% of galactosyltransferase activity compared to that of beta4Gal-T1, the enzyme present in milk. However, in the presence of alpha-LA, beta4Gal-T4 activity increases to nearly 100% of beta-4Gal-T1. This is in contrast to beta4Gal-T1, where alpha-LA enhances the transfer of Gal to glucose rather than to GlcNAc. By site-directed mutational analysis we have identified F280 and F360 in bovine beta4Gal-T1, the residues when mutated to Thr and Met, respectively, that are present at the corresponding positions in beta4Gal-T4, alters beta4Gal-T1 so that it exhibited beta4Gal-T4 property. Thus, it seems that these two Phe mutations may be primarily responsible for the basic characteristics of beta4Gal-T4.We have also cloned and expressed in E. coli the catalytic domain of bovine alpha-1,3-galactosyltransferase, the residues 80-368 of the enzyme, and obtained in soluble form a pure and active protein. The enzyme transfers galactose from UDP-alpha-D-galactose to N-acetylglucosamine (GlcNAc), with the ?retention? of the anomeric configuration at the C1 carbon atom of galactose, generating an alpha1-3-linkage. We have studied the activity of enzyme towards various acceptor substrates, including LacNAc, which is the natural substrate of the enzyme and correlated the activities with the preferred conformation of these substrates derived by molecular dynamics simulations.1) B. Ramakrishnan, P. S Shah, E. Boeggeman and P. K. Qasba. Glycoconjugate J. 16: S74, 19992) E. Boeggeman and P. K. Qasba. Glycobiology 8: Abstract # 141, 1998.
