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1,3-glucuronosyltransferase I (GlcAT-I)
1 UMR 7561 CNRS-Université Henri Poincaré Nancy I, Faculté de Médecine, 54505 Vand
uvre-lès-Nancy cedex, France
2 INSERM U602; Université de Paris XI, 94807 Villejuif, France
3 UMR 6005 CNRS, UFR Sciences, Université d'Orléans, 45067 Orléans cedex 02, France
4 UMR 7036 CNRS-Université Henri Poincaré Nancy I, Faculté des Sciences, 54505 Vanduvre-lès-Nancy cedex, France
(RECEIVED January 10, 2006; FINAL REVISION March 9, 2006; ACCEPTED March 9, 2006)
The 
1,3-glucuronosyltransferases are responsible for the completion of the protein–glycosaminoglycan linkage region of proteoglycans and of the HNK1 epitope of glycoproteins and glycolipids by transferring glucuronic acid from UDP-
-D-glucuronic acid (UDP-GlcA) onto a terminal galactose residue. Here, we develop phylogenetic and mutational approaches to identify critical residues involved in UDP-GlcA binding and enzyme activity of the human 1,3-glucuronosyltransferase I (GlcAT-I), which plays a key role in glycosaminoglycan biosynthesis. Phylogeny analysis identified 119 related 1,3-glucuronosyltransferase sequences in vertebrates, invertebrates, and plants that contain eight conserved peptide motifs with 15 highly conserved amino acids. Sequence homology and structural information suggest that Y84, D113, R156, R161, and R310 residues belong to the UDP-GlcA binding site. The importance of these residues is assessed by site-directed mutagenesis, UDP affinity and kinetic analyses. Our data show that uridine binding is primarily governed by stacking interactions with the phenyl group of Y84 and also involves interactions with aspartate 113. Furthermore, we found that R156 is critical for enzyme activity but not for UDP binding, whereas R310 appears less important with regard to both activity and UDP interactions. These results clearly discriminate the function of these two active site residues that were predicted to interact with the pyrophosphate group of UDP-GlcA. Finally, mutation of R161 severely compromises GlcAT-I activity, emphasizing the major contribution of this invariant residue. Altogether, this phylogenetic approach sustained by biochemical analyses affords new insight into the organization of the 1,3-glucuronosyltransferase family and distinguishes the respective importance of conserved residues in UDP-GlcA binding and activity of GlcAT-I.
Keywords: glucuronosyltransferase family; phylogeny; site-directed mutagenesis; enzyme kinetics; UDP-glucuronic acid binding site; glycosaminoglycan synthesis
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