High Resolution Structures of the Human ABO(H) Blood Group Enzymes in Complex with Donor Analogs Reveal That the Enzymes Utilize Multiple Donor Conformations to Bind Substrates in a Stepwise Manner
High Resolution Structures of the Human ABO(H) Blood Group Enzymes in Complex with Donor Analogs Reveal That the Enzymes Utilize Multiple Donor Conformations to Bind Substrates in a Stepwise Manner
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DOI：
10.1074/jbc.m115.682401
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发表时间：
2015-11-06
影响因子：
4.8
通讯作者：
Evans, Stephen V.
中科院分区：
文献类型：
--
作者：
Gagnon, Susannah M. L.;Meloncelli, Peter J.;Evans, Stephen V.
同型半乳糖基转移酶α-（1 -> 3）-N-乙酰氨基半乳糖基转移酶（GTA）和α-（1 -> 3）-半乳糖基转移酶（GTB）通过糖从活化供体转移到H抗原受体催化ABO（H）血型A和B抗原合成的最后一步。这些酶具有GT-A折叠类型，具有特征性移动的多肽环，其覆盖底物结合后的活性位点，并且尽管进行了深入的研究，但底物特异性和催化的许多方面仍不清楚。GTA、GTB和它们的嵌合体的结构已经被确定为与天然供体UDP-半乳糖、UDP-葡萄糖复合的1.55和1.39埃分辨率之间，并且试图克服与三维研究相关的常见问题之一，不可水解的供体类似物UDP-膦酰基-半乳糖（UDP-C-半乳糖）。虽然观察到供体的尿嘧啶部分保持恒定的位置，但糖部分存在于四种不同的构象中，从延伸到与催化相关的“折叠”构象，每种构象通过与酶的不同氢键合伙伴稳定。此外，几个结构显示出明确的证据，供体糖是无序超过两个观察到的构象，因此提供证据逐步插入到活性位点。虽然天然供体与酶复合时都能呈现下折构象，但UDP-C-Gal不能。尽管UDP-C-Gal被设计为与天然供体“等排”，但通过将差向异构体氧原子改变为碳所施加的结构上的微小差异似乎使酶不能以活性构象结合类似物，因此排除了其在GTA和GTB中作为底物模拟物的用途。
Homologous glycosyltransferases alpha-(1 -> 3)-N-acetylgalactosaminyltransferase (GTA) and alpha-(1 -> 3)-galactosyltransferase (GTB) catalyze the final step in ABO(H) blood group A and B antigen synthesis through sugar transfer from activated donor to the H antigen acceptor. These enzymes have a GT-A fold type with characteristic mobile polypeptide loops that cover the active site upon substrate binding and, despite intense investigation, many aspects of substrate specificity and catalysis remain unclear. The structures of GTA, GTB, and their chimeras have been determined to between 1.55 and 1.39 angstrom resolution in complex with natural donors UDP-Gal, UDP-Glc and, in an attempt to overcome one of the common problems associated with three-dimensional studies, the non-hydrolyzable donor analog UDP-phosphono-galactose (UDP-C-Gal). Whereas the uracil moieties of the donors are observed to maintain a constant location, the sugar moieties lie in four distinct conformations, varying from extended to the"tucked under" conformation associated with catalysis, each stabilized by different hydrogen bonding partners with the enzyme. Further, several structures show clear evidence that the donor sugar is disordered over two of the observed conformations and so provide evidence for stepwise insertion into the active site. Although the natural donors can both assume the tucked under conformation in complex with enzyme, UDP-C-Gal cannot. Whereas UDP-C-Gal was designed to be "isosteric" with natural donor, the small differences in structure imposed by changing the epimeric oxygen atom to carbon appear to render\ the enzyme incapable of binding the analog in the active conformation and so preclude its use as a substrate mimic in GTA and GTB.