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PEDS Advance Access published online on September 17, 2004
Protein Engineering Design and Selection, doi:10.1093/protein/gzh075
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Received July 13, 2004
Revised September 6, 2004
Accepted September 10, 2004

Article

Characterisation of the UDP-N-acetylgalactosamine binding domain of bovine polypeptide {alpha} N-acetylgalactosaminyltransferase T1

Stéphanie Duclos 1, Pedro Da Silva 1, Françoise Vovelle 1, Friedrich Piller 1, and Véronique Piller 1*

1 From the Centre de Biophysique Moléculaire, CNRS UPR 4301, affiliated with INSERM and the University of Orléans, F 45071 Orléans Cédex 2, France

* To whom correspondence should be addressed. E-mail: pillere{at}cnrs-orleans.fr.


  Abstract

UDP-GalNAc : polypeptide {alpha}N-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 {alpha}1,3galactosyltransferase and the human {alpha}1,3N-acetylgalactosaminyltransferase. A model of the ppGaNTase-T1 nucleotide-sugar binding site was built into which the UDP-GalNAc and the Mn2+ 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 Mn2+ 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.

Keywords: Glycosyltransferases; Polypeptide alpha N-acetylgalactosaminyltransferases; comparative molecular modelling; UDP-GalNAc; mutagenesis.
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