Fondeur-Gelinotte Magali, Lattard Virginie, Oriol Rafael, Mollicone Rosella, Jacquinet Jean-Claude, Mulliert Guillermo, Gulberti Sandrine, Netter Patrick, Magdalou Jacques, Ouzzine Mohamed, Fournel-Gigleux Sylvie
UMR 7561 CNRS-Université Henri Poincaré Nancy I, Faculté de Médecine, Vandoeuvre-lès-Nancy, France.
Protein Sci. 2006 Jul;15(7):1667-78. doi: 10.1110/ps.062089106.
The beta1,3-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-alpha-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 beta1,3-glucuronosyltransferase I (GlcAT-I), which plays a key role in glycosaminoglycan biosynthesis. Phylogeny analysis identified 119 related beta1,3-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 beta1,3-glucuronosyltransferase family and distinguishes the respective importance of conserved residues in UDP-GlcA binding and activity of GlcAT-I.
β1,3-葡糖醛酸基转移酶负责通过将UDP-α-D-葡糖醛酸（UDP-GlcA）中的葡糖醛酸转移到末端半乳糖残基上，来完成蛋白聚糖的蛋白质-糖胺聚糖连接区域以及糖蛋白和糖脂的HNK1表位。在此，我们开发了系统发育和突变方法，以鉴定参与人β1,3-葡糖醛酸基转移酶I（GlcAT-I）的UDP-GlcA结合和酶活性的关键残基，该酶在糖胺聚糖生物合成中起关键作用。系统发育分析在脊椎动物、无脊椎动物和植物中鉴定出119个相关的β1,3-葡糖醛酸基转移酶序列，这些序列包含8个保守肽基序和15个高度保守的氨基酸。序列同源性和结构信息表明，Y84、D113、R156、R161和R310残基属于UDP-GlcA结合位点。通过定点诱变、UDP亲和力和动力学分析来评估这些残基的重要性。我们的数据表明，尿苷结合主要由与Y84苯环的堆积相互作用决定，并且还涉及与天冬氨酸113的相互作用。此外，我们发现R156对酶活性至关重要，但对UDP结合不重要，而R310在活性和UDP相互作用方面似乎不太重要。这些结果清楚地区分了这两个预测与UDP-GlcA焦磷酸基团相互作用的活性位点残基的功能。最后，R161的突变严重损害了GlcAT-I的活性，强调了这个不变残基的主要贡献。总之，这种由生化分析支持的系统发育方法为β1,3-葡糖醛酸基转移酶家族的组织提供了新的见解，并区分了保守残基在UDP-GlcA结合和GlcAT-I活性中的各自重要性。
