ArticleProceedings of the National Academy of Sciences of the United States of America2014
Toward resolving the catalytic mechanism of dihydrofolate reductase using neutron and ultrahigh-resolution X-ray crystallography.
Article in Proceedings of the National Academy of Sciences of the United States of America, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 45 papers.
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Who cites it
45 citing papers in PubMed.
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- Evolution of split-protein technologies in virology: From mechanistic discovery and diagnostics to therapeutic promise.Journal of virus eradication · 2025Review
- Conformational flexibility of His200 enables catalytic activity in the T200H mutant of carbonic anhydrase II.Molecules and cells · 2025Article
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- Secondary Amine Catalysis in Enzyme Design: Broadening Protein Template Diversity through Genetic Code Expansion.Angewandte Chemie (International ed. in English) · 2024Article
- Perturbative diffraction methods resolve a conformational switch that facilitates a two-step enzymatic mechanism.Proceedings of the National Academy of Sciences of the United States of America · 2024Article
- Folate-producing bifidobacteria: metabolism, genetics, and relevance.Microbiome research reports · 2024Review
- X-ray-driven chemistry and conformational heterogeneity in atomic resolution crystal structures of bacterial dihydrofolate reductases.bioRxiv : the preprint server for biology · 2023Article
- Water Networks in Photosystem II Using Crystalline Molecular Dynamics Simulations and Room-Temperature XFEL Serial Crystallography.Journal of the American Chemical Society · 2023Article
- A conserved SH3-like fold in diverse putative proteins tetramerizes into an oxidoreductase providing an antimicrobial resistance phenotype.Philosophical transactions of the Royal Society of London. Series B, Biological sciences · 2023Article
- Modeling Ensembles of Enzyme Reaction Pathways with Hi-MSM Reveals the Importance of Accounting for Pathway Diversity.The journal of physical chemistry. B · 2022Article
- Native SAD phasing at room temperature.Acta crystallographica. Section D, Structural biology · 2022Article
- Dihydrofolate reductase, thymidylate synthase, and serine hydroxy methyltransferase: successful targets against some infectious diseases.Molecular biology reports · 2022Review
- Structure-guided functional studies of plasmid-encoded dihydrofolate reductases reveal a common mechanism of trimethoprim resistance in Gram-negative pathogens.Communications biology · 2022Article
- Hydrogen/Deuterium Exchange Behavior During Denaturing/Refolding Processes Determined in Tetragonal Hen Egg-White Lysozyme Crystals.Molecular biotechnology · 2022Article
- Computational protein design repurposed to explore enzyme vitality and help predict antibiotic resistance.Frontiers in molecular biosciences · 2022Article
- Pressure Adaptations in Deep-SeaBiology · 2021Article
- Switching an active site helix in dihydrofolate reductase reveals limits to subdomain modularity.Biophysical journal · 2021Article
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7 authors.
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Abstract
Dihydrofolate reductase (DHFR) catalyzes the NADPH-dependent reduction of dihydrofolate (DHF) to tetrahydrofolate (THF). An important step in the mechanism involves proton donation to the N5 atom of DHF. The inability to determine the protonation states of active site residues and substrate has led to a lack of consensus regarding the catalytic mechanism involved. To resolve this ambiguity, we conducted neutron and ultrahigh-resolution X-ray crystallographic studies of the pseudo-Michaelis ternary complex of Escherichia coli DHFR with folate and NADP(+). The neutron data were collected to 2.0-Å resolution using a 3.6-mm(3) crystal with the quasi-Laue technique. The structure reveals that the N3 atom of folate is protonated, whereas Asp27 is negatively charged. Previous mechanisms have proposed a keto-to-enol tautomerization of the substrate to facilitate protonation of the N5 atom. The structure supports the existence of the keto tautomer owing to protonation of the N3 atom, suggesting that tautomerization is unnecessary for catalysis. In the 1.05-Å resolution X-ray structure of the ternary complex, conformational disorder of the Met20 side chain is coupled to electron density for a partially occupied water within hydrogen-bonding distance of the N5 atom of folate; this suggests direct protonation of substrate by solvent. We propose a catalytic mechanism for DHFR that involves stabilization of the keto tautomer of the substrate, elevation of the pKa value of the N5 atom of DHF by Asp27, and protonation of N5 by water that gains access to the active site through fluctuation of the Met20 side chain even though the Met20 loop is closed.
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