title: Structural basis for the inhibition of human 5,10-methenyltetrahydrofolate synthetase by N10-substituted folate analogues.
abstract: 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.
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
authors.forename: Dong
authors.initials: D
authors.lastname: Wu
authors.forename: Yang
authors.initials: Y
authors.lastname: Li
authors.forename: Gaojie
authors.initials: G
authors.lastname: Song
authors.forename: Chongyun
authors.initials: C
authors.lastname: Cheng
authors.forename: Rongguang
authors.initials: R
authors.lastname: Zhang
authors.forename: Andrzej
authors.initials: A
authors.lastname: Joachimiak
authors.forename: Neil
authors.initials: N
authors.lastname: Shaw
authors.forename: Zhi-Jie
authors.initials: ZJ
authors.lastname: Liu
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D019731:Carbon-Nitrogen Ligases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D005492:Folic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D000255:Adenosine Triphosphate; D005492:Folic Acid; D019731:Carbon-Nitrogen Ligases; C021557:5,10-methenyltetrahydrofolate synthetase