Wu Dong, Li Yang, Song Gaojie, Cheng Chongyun, Zhang Rongguang, Joachimiak Andrzej, Shaw Neil, Liu Zhi-Jie
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
Cancer Res. 2009 Sep 15;69(18):7294-301. doi: 10.1158/0008-5472.CAN-09-1927. Epub 2009 Sep 8.
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.
5,10-亚甲基四氢叶酸合成酶（MTHFS）调节一碳代谢网络中的碳流，该网络为细胞的生长和增殖提供必需成分。已证明抑制人MCF-7乳腺癌细胞中的MTHFS会阻止细胞生长。人MTHFS（hMTHFS）三维结构的缺失阻碍了候选药物的合理设计和优化。在此，我们报告了天然hMTHFS、hMTHFS与ADP的二元复合物、与N5-亚胺基磷酸反应中间体结合的hMTHFS以及hMTHFS的酶-产物复合物的结构。我们在晶体结构中首次捕获的N5-亚胺基磷酸揭示了hMTHFS识别底物所采用的独特策略，并为酶活性调节提供了结构基础。N10-取代叶酸类似物的结合使Y152位于连接ATP结合位点与底物结合口袋的通道中间，阻止了γ-磷酸进行亲核攻击的定位。以hMTHFS的结构为指导，我们通过诱变探究了活性位点周围残基在催化中的作用。hMTHFS结构与诱变数据共同呈现了MTHFS活性位点、底物特异性决定因素以及对hMTHFS抑制机制的新见解。
