Crystal structures of the bovine β4galactosyltransferase catalytic domain and its complex with uridine diphosphogalactose
Crystal structures of the bovine β4galactosyltransferase catalytic domain and its complex with uridine diphosphogalactose
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DOI：
10.1093/emboj/18.13.3546
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发表时间：
1999-07-01
期刊：
影响因子：
11.4
通讯作者：
Bourne, Y
中科院分区：
文献类型：
--
作者：
Gastinel, LN;Cambillau, C;Bourne, Y
β 1,4-半乳糖转移酶T1 （β 4Gal-T1, EC 2.4.1.90/38）是一种高尔基常住膜结合酶，它将尿苷二磷酸半乳糖中的半乳糖转移到末端的β - n -乙酰氨基葡萄糖胺残基上，形成糖蛋白和鞘糖脂中存在的多- n -乙酰氨基乳糖胺核心结构。在哺乳动物中，β - 4Gal-T1与α -乳蛋白（一种结构上与溶菌酶同源的蛋白质）结合产生乳糖，β - Gal-T1是使用不同类型的糖蛋白和糖脂作为底物的β - 4半乳糖转移酶大家族的成员。在此，我们对重组牛β 4Gal-T1在存在和不存在底物尿苷二磷酸半乳糖的情况下的晶体结构进行了求解和细化到2.4埃的分辨率。无牛底物β - 4Gal-T1催化结构域的晶体结构呈现出一个新的褶皱，由一个单一的锥形结构域组成，其底部有一个大的开口袋。在底物结合复合物中，袋状结构包含与尿苷二磷酸半乳糖相互作用的残基。该复合物的结构包含底物尿苷二磷酸部分的清晰电子密度区域，其β -磷酸基团通过与保守残基（包括Asp252ValAsp254飞虫）的氢键接触而稳定，这些结果有助于解释工程β 4Gal-T1点突变。他们提出了一种可能参与半乳糖转移的机制，并能够识别参与α -乳清蛋白相互作用的关键氨基酸。
beta 1,4-galactosyltransferase T1 (beta 4Gal-T1, EC 2.4.1.90/38), a Golgi resident membrane-bound enzyme, transfers galactose from uridine diphosphogalactose to the terminal beta-N-acetylglucosamine residues forming the poly-N-acetylactosamine core structures present in glycoproteins and glycosphingolipids. In mammals, beta 4Gal-T1 binds to alpha-lactalbumin, a protein that is structurally homologous to lyzozyme, to produce lactose, beta Gal-T1 is a member of a large family of homologous beta 4galactosyltransferases that use different types of glycoproteins and glycolipids as substrates. Here we solved and refined the crystal structures of recombinant bovine beta 4Gal-T1 to 2.4 Angstrom resolution in the presence and absence of the substrate uridine diphosphogalactose. The crystal structure of the bovine substrate-free beta 4Gal-T1 catalytic domain showed a new fold consisting of a single conical domain with a large open pocket at its base. In the substrate-bound complex, the pocket encompassed residues interacting with uridine diphosphogalactose. The structure of the complex contained clear regions of electron density for the uridine diphosphate portion of the substrate, where its beta-phosphate group was stabilized by hydrogen-bonding contacts with conserved residues including the Asp252ValAsp254 moth, These results help the interpretation of engineered beta 4Gal-T1 point mutations, They suggest a mechanism possibly involved in galactose transfer and enable identification of the critical amino acids involved in alpha-lactalbumin interactions.