Active site studies of bovine alpha 1 -> 3-galactosyltransferase and its secondary structure prediction

The catalytic domain of bovine alpha 1 --> 3-galactosyltransferase (alpha 3 GalT), residues 80-368, have been cloned and expressed, in Escherichia coli . Using a sequential purification protocol involving a Ni2+ affinity column followed by a UDP-hexanolamine affinity column, we have obtained a pure an d active protein from the soluble fraction which catalyzes the transfer of galactose (Gal) from UDP-Gal to N-acetyllactosamine (LacNAc) with a specifi c activity of 0.69 pmol/min/ng. The secondary structural content of alpha 3 GalT protein was analyzed by Fourier transform infrared (FTIR) spectroscopy , which shows that the enzyme has about 35% beta-sheet and 22% alpha-helix. This predicted secondary structure content by FTIR spectroscopy was used i n the protein sequence analysis algorithm, developed by the Biomolecular En gineering Research Center at Boston University and Tasc Inc., for the assig nment of secondary structural elements to the amino acid sequence of alpha 3GalT. The enzyme appears to have three major and three minor helices and f ive sheet-like structures. The studies on the acceptor substrate specificit y of the enzyme, alpha 3GalT, show that in addition to LacNAc, which is the natural substrate, the enzyme accepts various other disaccharides as subst rates such as lactose and Gal derivatives, beta-O-methylgalactose and beta- D-thiogalactopyranoside, albeit with lower specific activities. There is an absolute requirement for Gal to be at the non-reducing end of the acceptor molecule which has to be beta 1 --> 4-linked to a second residue that can be more diverse in structure. The kinetic parameters for four acceptor mole cules were determined. Lactose binds and functions in a similar way as LacN Ac. However. beta-O-methylgalactose and Gal do not bind as tightly as LacNA c or lactose, as their K-ia and K-A values indicate, suggesting that the se cond monosaccharide is critical for holding the acceptor molecule in place. The 2' and 4' hydroxyl groups of the receiving Gal moiety are important in binding. Even though there is large structural variability associated with the second residue of the acceptor molecule, there are constraints which d o not allow certain Gal-R sugars to be good acceptors for the enzyme. The b eta 1 --> 4-linked residue at the second position of the acceptor molecule is preferred, but the interactions between the enzyme and the second residu e are likely to be non-specific. (C) 2000 Elsevier Science B.V. All rights reserved.