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Structural Basis for the Inhibition of Human 5,10-Methenyltetrahydrofolate Synthetase by N10-Substituted Folate Analogues

¹ National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China and ² Structural Biology Center, Argonne National Laboratory, Argonne, Illinois

Requests for reprints: Zhi-Jie Liu or Neil Shaw, National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China. Phone: 86-10-64857988; Fax: 86-10-64888426; E-mail: zjliu{at}ibp.ac.cn or neilshaw{at}moon.ibp.ac.cn.

Key Words: antifolate • cancer • 5,10-methenyltetrahydrofolate synthetase • one-carbon metabolic network • X-ray crystallography

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 -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. [Cancer Res 2009;69(18):7294–301]