Cysteine-to-serine mutants dramatically reorder the active site of human ABO(H) blood group B glycosyltransferase without affecting activity: structural insights into cooperative substrate binding.
Cysteine-to-serine mutants dramatically reorder the active site of human ABO(H) blood group B glycosyltransferase without affecting activity: structural insights into cooperative substrate binding.
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半胱氨酸到丝氨酸突变体显着重新排序人 ABO(H) 血型 B 糖基转移酶的活性位点而不影响活性：对协同底物结合的结构见解。
DOI：
10.1016/j.jmb.2010.07.036
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发表时间：
2010
影响因子：
5.6
通讯作者：
S. V. Evans
中科院分区：
文献类型：
--
作者：
B. Schuman;M. Persson;R. Landry;R. Polakowski;J. Weadge;N. Seto;S. Borisova;M. Palcic;S. V. Evans
GT-A折叠型糖基转移酶结构的共同特征是移动的多肽环，已观察到其参与底物识别并在底物结合时包围活性位点。这是人ABO（H）血型B糖基转移酶GTB的情况，其中氨基酸残基177-195在未配体状态下比在完全配体状态下显示出显著更高水平的紊乱。在1.93至1.40 Å的分辨率范围内对突变酶GTB/C80 S/C196 S和GTB/C80 S/C196 S/C209 S进行的结构研究显示出相反的趋势，其中无配体结构显示出在与底物结合时丢失的移动的环残基几乎完全有序。在突变体结构的配体状态下，虽然观察到供体分子的UDP部分在预期位置结合，但观察到半乳糖部分以与野生型嵌合结构观察到的构象显著不同的构象结合。虽然这将预期阻碍催化周转，但转移反应的动力学在很大程度上不受影响。这些结构表明，酶以与主要溶液旋转异构体更相似的构象结合供体，并促进其旋转成有催化活性的形式。此外，通过防止活性位点封闭，这些结构提供了最近观察到的底物结合的协同性的基础。最后，C80 S的突变在酶二聚体界面处引入完全占据的UDP结合位点，观察到其依赖于H抗原受体类似物的结合。
A common feature in the structures of GT-A-fold-type glycosyltransferases is a mobile polypeptide loop that has been observed to participate in substrate recognition and enclose the active site upon substrate binding. This is the case for the human ABO(H) blood group B glycosyltransferase GTB, where amino acid residues 177–195 display significantly higher levels of disorder in the unliganded state than in the fully liganded state. Structural studies of mutant enzymes GTB/C80S/C196S and GTB/C80S/C196S/C209S at resolutions ranging from 1.93 to 1.40 Å display the opposite trend, where the unliganded structures show nearly complete ordering of the mobile loop residues that is lost upon substrate binding. In the liganded states of the mutant structures, while the UDP moiety of the donor molecule is observed to bind in the expected location, the galactose moiety is observed to bind in a conformation significantly different from that observed for the wild-type chimeric structures. Although this would be expected to impede catalytic turnover, the kinetics of the transfer reaction are largely unaffected. These structures demonstrate that the enzymes bind the donor in a conformation more similar to the dominant solution rotamer and facilitate its gyration into the catalytically competent form. Further, by preventing active-site closure, these structures provide a basis for recently observed cooperativity in substrate binding. Finally, the mutation of C80S introduces a fully occupied UDP binding site at the enzyme dimer interface that is observed to be dependent on the binding of H antigen acceptor analog.
α-1,3-半乳糖基转移酶在催化和特异性中单个酶-底物相互作用的作用。
DOI：
10.1021/bi035430r
发表时间：
2003
期刊：
Biochemistry.
影响因子：
--
作者：
Zhang,Yingnan;Swaminathan,GJawahar;Deshpande,Ashlesha;Boix,Ester;Natesh,Ramanathan;Xie,Zhihong;Acharya,KRavi;Brew,Keith
通讯作者：
Brew,Keith
影响因子：
4.8
作者：
Ramakrishnan, B;Shah, PS;Qasba, PK
通讯作者：
Qasba, PK