Structural basis for the inhibition of human 5,10-methenyltetrahydrofolate synthetase by N10-substituted folate analogues.
Structural basis for the inhibition of human 5,10-methenyltetrahydrofolate synthetase by N10-substituted folate analogues.
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DOI：
10.1158/0008-5472.can-09-1927
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发表时间：
2009-09
期刊：
影响因子：
11.2
通讯作者：
Dong Wu;Yang Li;Gaojie Song;Chongyun Cheng;Rong-guang Zhang;A. Joachimiak;N. Shaw;Zhi-jie Liu
中科院分区：
文献类型：
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作者：
Dong Wu;Yang Li;Gaojie Song;Chongyun Cheng;Rong-guang Zhang;A. Joachimiak;N. Shaw;Zhi-jie Liu
5，10-亚甲基四氢叶酸合成酶（MTHFS）通过一碳代谢网络调节碳的流动，为细胞的生长和增殖提供必需的组分。在人MCF-7乳腺癌细胞中抑制MTHFS已显示出阻止细胞生长。人MTHFS（hMTHFS）三维结构的缺乏阻碍了候选药物的合理设计和优化。在这里，我们报告的天然hMTHFS的结构，一个二元复合物的hMTHFS与ADP，hMTHFS绑定的N5-磷酸亚胺反应中间体，和酶的产物复合物的hMTHFS。N5-磷酸亚胺首次在我们的晶体结构中被捕获，揭示了hMTHFS识别底物的独特策略，并为酶活性的调节提供了结构基础。N10取代的叶酸类似物的结合将Y152置于连接ATP结合位点与底物结合口袋的通道的中间，排除了用于亲核攻击的γ-磷酸的定位。使用hMTHFS的结构作为指导，我们已经探索了周围的活性位点的突变催化残基的作用。的hMTHFS结构和诱变数据的合奏产生的MTHFS活性位点，底物特异性的决定因素，和新的见解的抑制hMTHFS的机制的连贯画面。
5,10-Methenyltetrahydrofolate synthetase (MTHFS) regulates the flow of carbon through the one-carbon metabolic network, which supplies essential components for the growth and proliferation of cells. Inhibition of MTHFS in human MCF-7 breast cancer cells has been shown to arrest the growth of cells. Absence of the three-dimensional structure of human MTHFS (hMTHFS) has hampered the rational design and optimization of drug candidates. Here, we report the structures of native hMTHFS, a binary complex of hMTHFS with ADP, hMTHFS bound with the N5-iminium phosphate reaction intermediate, and an enzyme-product complex of hMTHFS. The N5-iminium phosphate captured for the first time in our crystal structure unravels a unique strategy used by hMTHFS for recognition of the substrate and provides structural basis for the regulation of enzyme activity. Binding of N10-substituted folate analogues places Y152 in the middle of the channel connecting ATP binding site with the substrate binding pocket, precluding the positioning of gamma-phosphate for a nucleophilic attack. Using the structures of hMTHFS as a guide, we have probed the role of residues surrounding the active site in catalysis by mutagenesis. The ensemble of hMTHFS structures and the mutagenesis data yield a coherent picture of the MTHFS active site, determinants of substrate specificity, and new insights into the mechanism of inhibition of hMTHFS.