Characterization of the UDP-N-acetylgalactosamine binding domain of bovine polypeptide N-acetylgalactosaminyltransferase T1

S. Duclos, P. Da Silva, F. Vovelle, F. Piller, V. Piller
2004 Protein Engineering Design & Selection  
UDP-GalNAc:polypeptide aN-acetylgalactosaminyltransferases (ppGaNTases) transfer GalNAc from UDP-GalNAc to Ser or Thr. Structural features underlying their enzymatic activity and their specificity are still unidentified. In order to get some insight into the donor substrate recognition, we used a molecular modelling approach on a portion of the catalytic site of the bovine ppGaNTase-T1. Fold recognition methods identified as appropriate templates the bovine a1,3galactosyltransferase and the
more » ... n a1,3N-acetylgalactosaminyltransferase. A model of the ppGaNTase-T1 nucleotide-sugar binding site was built into which the UDP-GalNAc and the Mn 2+ cation were docked. UDP-GalNAc fits best in a conformation where the GalNAc is folded back under the phosphates and is maintained in that special conformation through hydrogen bonds with R193. The ribose is found in van der Waals contacts with F124 and L189. The uracil is involved in a stacking interaction with W129 and forms a hydrogen bond with N126. The Mn 2+ is found in coordination both with the phosphates of UDP and the DXH motif of the enzyme. Amino acids in contact with UDP-GalNAc in the model have been mutated and the corresponding soluble forms of the enzyme expressed in yeast. Their kinetic constants confirm the importance of these amino acids in donor substrate interactions.
doi:10.1093/protein/gzh075 pmid:15377782 fatcat:kff75vtskjc27mpa6phh2zdwi4