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1.5.1.3: dihydrofolate reductase

This is an abbreviated version!
For detailed information about dihydrofolate reductase, go to the full flat file.

Word Map on EC 1.5.1.3

Reaction

5,6,7,8-tetrahydrofolate
+
NADP+
=
7,8-dihydrofolate
 +
NADPH
 +
H+

Synonyms

5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, 7,8-dihydrofolate reductase, At2g16370, At4g34570, bifunctional dihydrofolate reductase-thymidylate synthase, bifunctional TS-DHFR, BmDHFR, dehydrogenase, tetrahydrofolate, DFR-TS, DFR1, DfrA, DfrB, DHFR, DHFR type IIIC, DHFR-TS, DHFR-TS1, DHFR-TS2, DHFR2, DHFRL1, DHFRLS, dihydrofolate reductase, dihydrofolate reductase-like, dihydrofolate reductase-thymidylate synthase, dihydrofolate reductase:thymidylate synthase, dihydrofolic acid reductase, dihydrofolic reductase, EC 1.5.1.4, ecDHFR, folA, folA3, folic acid reductase, folic reductase, FolM, hDHFR, hDHFR-1, hDHFR-2, HjDHFR, hvDHFR1, hvDHFR2, LAU_0427, LBRM_06_0830, mDHFR, mjDHFR, More, myDHFR, NADPH-dihydrofolate reductase, pcDHFR, PKNH_0509600, ppDHFR, pteridine reductase, pteridine reductase:dihydrofolate reductase, PTR2, R-plasmid-encoded dihydrofolate reductase, R67 DHFR, R67 dihydrofolate reductase, reductase, dihydrofolate, S3DHFR, Smdhfr, Smp 175230, spDHFR, svDHFR, tcptr1, tetrahydrofolate dehydrogenase, THY-1, THY-2, thymidylate synthase-dihydrofolate reductase, thymidylate synthetase-dihydrofolate reductase, Trimethoprim resistance protein, TS-DHFR, WUBG_00817

ECTree

     1 Oxidoreductases
         1.5 Acting on the CH-NH group of donors
             1.5.1 With NAD+ or NADP+ as acceptor
                1.5.1.3 dihydrofolate reductase

Crystallization

Crystallization on EC 1.5.1.3 - dihydrofolate reductase

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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
enzyme in ternary complex with NADPH and inhibitor methotrexate, soaking of crystals in 0.1 M Bis-Tris, pH 5.5, 0.2 M CaCl2 and 22.5% w/v PEG 3350 supplemented with 5 mM beta-NADPH at 4°C for 8 h, cryoprotection with 20% v/v glycerol and 5 mM beta-NADPH, X-ray diffraction structure determination and analysis at 2.3 A resolution
in complex with methotrexate, at 2.4 A resolution. Modeling of inhibitor 5-nitro-6-methylamino-isocytosine into the active site reveals four potential hydrogen bonds
purified recombinant His-tagged enzyme in complex with inhibitors RAB1 and trimethoprim, DHFR-RAB1 cocrystals grow within 1 to 2 days at 22°C in a sitting-drop format from a 30 mg/ml protein solution mixed 1:1 with a well solution containing 12% PEG 3350, 0.2 M CaCl2, 0.1 M MES, pH 5.8, and 3% glycerol. DHFR-TMP cocrystals grow within 10 days at 22°C in a sitting-drop format from a 42.5 mg/ml protein solution mixed 1:1 with a well solution containing 13% PEG 3350, 0.2 M CaCl2, 0.1 M MES, pH 5.45, and 1% ethanol, X-ray diffraction structure determination and analysis at 2.3 A resolution
purified recombinant mutant V77A/I130M/I138V enzyme in complex with inhibitor 2,4-diamino-5-[3-(2,5-dimethoxyphenyl)prop-1-ynyl]-6-ethylpyrimidine, hanging-drop vapor diffusion in 25% w/v PEG 10,000, 0.1 M MES, pH 6.5, at an equal ratio of protein to crystallization solution. Microseeding is used to obtain isolated crystals in 10% w/v PEG 10000 and 0.1 M MES, pH 6.5, at a protein concentration of 10 mg/ml, improved by 15% ethylene glycol. X-ray diffraction structure determination and analysis at 2.5 A resolution
wild type and mutant enzymes bound to trimethoprim and propargyl-linked inhibitors, hanging drop vapor diffusion method, using 22.5% (w/v) PEG 10000, 0.1 M MES, pH 6.75. Microseeding is used to obtain isolated crystals in 12% (w/v) PEG 10000 and 0.1 MES, pH 6.7
comparison of the backbone conformation in crystal structures, detection of mutational insertions
-
co-crystallization of the purified enzyme with inhibitor 2-amino-4-oxo-4,7-dihydro-pyrrolo[2,3-d]pyrimidine-methyl-phenyl-L-glutamic acid and FdUMP in the TS site and NADPH and methotrexate in the DHFR site, X-ray diffraction structure determination and analysis at 3.45 A resolution, PDB ID 4Q0D
purified recombinant detagged enzyme in apoform or complex with inhibitor RAB, sitting drop vapor diffusion method, for the apoenzyme, mixing of 800 nl of 16.5 mg/ml protein in 20 mM Tris, pH 8.0, 150 mM NaCl, and 2 mM DTT, with 800 nl of well solution containing 5% PEG 3350 and 1.5 M ammonium citrate dibasic, pH 7.0, 1 week at room temperature, crystals complexed with RAB are grown from 1.1 M ammonium tartrate dibasic, pH 7.0, at 4°C in 2-3 months, X-ray diffraction structure determination and analysis at 2.1-2.3 A resolution, molecular replacement and modeling using DHFR structure from Bacillus stearothermophilus, PDB ID 1ZDR, as search model
2.1 A resolution neutron structure of a pseudo-Michaelis complex determined at acidic pH, direct observation of the catalytic proton and its parent solvent molecule
analysis of higher energy conformational substrates by NMR relaxation dispersion. The maximum hydride transfer and steady-state turnover rates are governed by the dynamics of transitions between ground and excited states of the intermediates. Model of conformational changes during the catalytic cycle
binding to trimethoprim, structure analysis
-
comparison of temperature dependence of dynamics between Geobacillus stearothermophilus and Escherichia coli enzymes using elastic coherent scattering. The Geobacillus' enzyme has a significantly broader distribution and slightly larger amplitudes of the atomic mean-square displacements extracted from the dynamic structure factor
crystal structure of complex with methotrexate
-
crystal structure of seleno- and telluromethionine-containing enzyme in complex with methotrexate and CaCl2 by vapour diffusion hanging drop method, X-ray analysis with synchotron and rotating anode generator as X-ray source
in complex with inhibitor 1,4-phenylenebis(methylene) bis(N-[amino(imino)methyl](imidothiocarbamate)) and NADPH. Inhibitor exploits a unique binding surface in loop M20
in the excited state of the enzyme:THF:NADPH product release complex, the reduced nicotinamide ring of the cofactor transiently enters the active site where it displaces the pterin ring of the THF product. The p-aminobenzoyl-L-glutamate tail of THF remains weakly bound in a widened binding cleft
microsecond compaction dynamics study of the folding. A significant collapse of the radius of gyration from 30 A to 23.2 A occurs within 0.3 sec after the initiatiation of refolding by a urea dilution jump. the subsequent folding occurs on a considerably longer time scale. Experimental data may best be explained by the specific hydrophobic collapse model. The folding trajectory of the protein is located between those of alpha-helical and beta-sheet proteins
modeling of complexes with inhibitors hydrochlorothiazide and indapamide. Compared to the DHFR-ethacrynic acid complex, DHFR-sulphadiazine, DHFR-indapamide, and DHFR-hydrochlorothiazide complexes have higher negative binding energies. The compounds may act similar to antibiotics from the perspective of binding with DHFR
mutant M1A/M16N/M20L/M42Y/M92F/C85A/C152S, increased structural flexibility and an increased size of the N-(p-aminobenzoyl)-L-glutamate binding cleft
self-organized polymer model to monitor the kinetics of closed to occluded and reverse transitions. During the closed to occluded transition, coordinated changes in a number of residues in the loop domain enable the M20 loop to slide along the alpha-helix in the adenosine-binding domain. Sliding is triggered by pulling of the M20 loop by the betaG-betaH loop and the pushing action of the betaG-betaH loop. The residues that facilitate the M20 loop motion are part of the network of residues that transmit allosteric signals during the close to occluded transition. Replacement of M16 and G121 by a disulfide cross-link impedes that transition. The order of events in the occluded to closed transition is not the reverse of the forward transition. The contact E18-S49 in the occluded structure persists until the sliding of the M20 loop is nearly complete
structure of plasmid-encoded R67 isoform bound to NADP+, at 1.15 A resolution. NADP+ assumes an extended conformation with the nicotinamide ring bound near the center of the active site pore, the ribose and diphospate group extending toward the outer pore. the ribonicotinamide exhibits anti conformation, and the coenzyme displayes symmetrical binding mode with several water-mediated hydrogen bonds
structure of ternary complex with NADPH and methotrexate
-
structures of protein crystallized in varying ionic strengths. High ionic strengths (0.75/1.5M) can preferentially stabilize the loop in closed/occluded conformations
study on enzyme ternary complex with substrate analogue folates and oxidized NADP+ cofactor using NMR relaxation methods. Conformational exchanges of protein between a ground state with closed conformation of active site loops and an excited state with loops in occluded conformation. Fluctuations include motions of the nicotinamide ring of the cofactor into and out of the active site
ternary complex of isoform R67 with dihydrofolate and NADPH, resolved to 1.26 A. In the catalytic complex, the polar backbone atoms of two symmetry-related I68 residues provide recognition motifs that interact with the carboxamide on the nicotinamide ring, and the N3-O4 amide function on the pteridine ring. This set of interactions orients the aromatic rings of substrate and cofactor in a relative endo geometry in which the reactive centers are held in close proximity. Additionally, a central, hydrogen-bonded network consisting of two pairs of Y69-Q67/Q67-Y69 residues provides a tight interface, which holds the substrates in place in an orientation conductive to hydride transfer
use of carbon-deuterium bonds as probes of proteins. The stretching absorption frequency of (methyl-d3) methionine carbon-deuterium bonds shows an approximately linear dependence on solvent dielectric. Characterization of the IR absorptions at residues Met16 and Met20, within the catalytically important Met20 loop, and Met42, which is located within the hydrophobic core of the enzyme. The carbon-deuterium bonds tare sensitive to their local protein environment and dihydrofolate reductase is electrostatically and dynamically heterogeneous
-
crystal structure, complex with 2,4-diamino-5,6-dihydro-6,6-dimethyl-5-(4'-methoxyphenyl)-s-triazine
-
structure of ternary complex with NADPH and trimethoprim, model development
-
comparison of temperature dependence of dynamics between Geobacillus stearothermophilus and Escherichia coli enzymes using elastic coherent scattering. The Geobacillus' enzyme has a significantly broader distribution and slightly larger amplitudes of the atomic mean-square displacements extracted from the dynamic structure factor
-
NMR-derived binary hvDHFR1:folate complex structure is presented to explore any possible conformational changes that occur upon binding folate
the crystal structure is determined at 2.6 A resolution. The structure is in the apo state, with an open conformation of the active-site gully
enzyme in ternary complex with NADPH and inhibitor (Z)-2,4-diamino-5-[2-(2'-methoxyphenyl)-propenyl]-furo[2,3-d]pyrimidine, protein is incubated with a 10:1 molar excess of NADPH and the inhibitor for one h over ice prior to crystallization using the hanging drop vapor diffusion method, 0.008 ml of protein solution containing 100 mM K2HPO4, pH 6.9, and 30% saturated ammonium sulfate, over a reservoir solution of 100 mM K2HPO4, pH 6.9, and 60% saturated ammonium sulfate with 3% v/v ethanol, several days, X-ray diffraction structure determination and analysis at 1.4-1.7 A resolution
enzyme mutant Q35S/N64S in complex with NADPH and inhibitor N-(4-[(2-amino-6-ethyl-4-oxo-3,4-dihydrothieno[2,3-d ]pyrimidin-5-yl)thio]benzoyl)-L-glutamic acid, and enzyme mutant Q35K in complex with NADPH and inhibitor N-{4-[(2-amino-6-methyl-4-oxo-3,4-dihydrothieno[2,3-d ]pyrimidin-5-yl)thio]benzoyl}-L-glutamic acid, X-ray diffraction structure determination and analysis at 1.2-1.5 A resolution
no crystallographic data available, but model-based structure analysis
-
purified enzyme in ternary complex with inhibitors N6-methyl-N6-(4-isopropylphenyl)pyrido[2,3-d]pyrimidine-2,4,6-triamine or N6-methyl-N6-(3,4,5-trifluorophenyl)pyrido[2,3-d]pyrimidine-2,4,6-triamine, and NADPH, hanging drop vapor diffusion method, mixing of 7.9 mg/ml protein in 100 mM K2HPO4, pH 6.9, and 30% saturated ammonium sufate, and a tenfold excess of NADPH and inhibitor, with reservoir solution consisting of 100 mM K2HPO4, pH 6.9, 60% saturated ammonium sulfate, and 3% v/v ethanol, X-ray diffraction structure determination and analysis at 1.54 A resolution, modeling
purified recombinant detagged wild-type enzyme and mutants Q35K and Q35K/N64F in complex with inhibitor 2,4-diamino-6-[(2',5'-dichloro anilino)methyl]pyrido[2,3-d]pyrimidine, hanging drop vapour diffusion method, mixing of 6.6-9.8 mg/ml protein in 100 mM K2HPO4, pH 6.9, and 30% saturated (NH4)2SO4 with a 0:1 molar excess of NADPH and inhibitor, and with reservoir solution containing 100 mM K2HPO4, pH 6.9, 60-64% saturated (NH4)2SO4, and 3% v/v ethyl alcohol, 0.01 ml drops, 14°C X-ray diffraction structure determination and analysis at 2.2 A resolution, molecular replacement methods using the coordinates for human DHFR, PDB ID 1U72, modeling
purified recombinant wild-type and mutant enzymes Q35K, Q35S/N64F, and Q35S/N64S in complex with NADPH and inhibitor PY957, hanging drop vapor diffusion method using, 0.010 ml protein solution containing 4.2 mg/ml for the single mutant protein and 6.6 mg/ml for the double mutant proteins in 100 mM K2HPO4, pH 6.9, with 30% saturated ammonium sulfate with a 10:1 molar excess of NADPH and PY957, are mixed with reservoir solution containing 100 mM K2HPO4, pH 6.9, and 60% saturated ammonium sulfate with 3% v/v ethanol, 3 days at 14°C, X-ray diffraction structure determination and analysis at 1.2-2.0 A resolution
structure-based three-dimensional quantitative structure-activity relationship 3D-QSAR approach to predict the biochemical activity for inhibitors of Trypanosoma cruzi dihydrofolate reductase-thymidylate synthase. Crystal structures of complexes of the enzyme with eight different inhibitors of the DHFR activity together with the structure in the substrate-free state have been used to validate and refine docking poses of ligands
wild-type and mutant L22R in ternary complex with methotrexate and NADPH, comparison with mouse anologues. Active site of mouse enzyme is larger than that of human enzyme
-
purified recombinant His-tagged enzyme in complex with NADPH and inhibitor 3 or 4, sitting drop vapour diffusion method, mixing of 15 mg/ml protein with 1 mM NADPH, and 1 mM inhibitor 4 and concentration to 24 mg/ml protein, addition of reservoir solution containing 100 mM cacodylate, pH 7.4, 150 mM sodium acetate, 25% w/v PEG 8000 in a 1:2 ratio to 0.01 ml sitting drops, equilibration against 1 ml reservoir solution with additional 1 mM (final concentration) L-glutathione, or mixing of 15 mg/ml protein with 1 mM NADPH, and 1 mM inhibitor 3 and concentration to 24 mg/ml protein, addition of reservoir solution, containing 100 mM cacodylate, pH 7.4, 125 mM sodium acetate, 25% w/v PEG 8000, and 11 mM calcium chloride, in a 1:2 ratio to 0.01 ml sitting drops, equilibration against 1 ml reservoir solution with additional 11 mM (final concentration) betaine hydrochloride, both 1-5 days at 4°C, X-ray diffraction structure determination and analysis at 1.76 A resolution, molecular replacement
structure of ternary complex with NADPH and methotrexate
-
structure of ternary complex with NADPH and phenyltriazine
-
holo and ternary complexes with NADPH and the inhibitor 2,4-diamino-6-(2-hydroxydibenz[b,f]azepin-5-yl)methylpteridine, to 1.9 and 1.4 A resolution, respectively. Modeling data of inhibitor 4-([5-[(2,4-diaminopteridin-6-yl)methyl]-5H-dibenzo[b,f]azepin-2-yl]oxy)butane-1,1-diol in the active site indicate that binding would require ligand-induced conformational changes to the enzyme for the inhibitor to fit or that the inhibitor side-chain would have to adopt an alternative binding mode to that observed for similar inhibitors. The complexes have a decreased active-site volume compared with complexes of the enzyme from Pneumocystis carinii
wild-type and mutant L22R in ternary complex with methotrexate and NADPH, comparison with human anologues. Active site of mouse enzyme is larger than that of human enzyme
-
binding mode analysis and docking approaches of inhibitors, and comparison between human and Mycobacterium tuberculosis enzyme. Presence of empty spaces around the 2,4-diamonodeazapteridine and N10-phenyl rings of inhibitors in the Mycobacterium tuberculosis enzyme active site that are not found in the human structures
-
in complex with inhibitor isoniazid, acyclic 4R isonicotinoyl-NADP adduct binds to and inhibits enzyme
structures of dihydrofolate reductase in complex with antifolates, pyrimethamine, cycloguanil, diaverdine and pemetrexed, and with substrate dihydrofolate. The structures of all complexes are obtained in the closed-conformation state of the enzyme and motion in key regions of the substrate-binding site and different binding modes of the ligands are observed
CoMFA and quantum chemical calculations studies on pyrimethamine derivatives active against quadruple mutant N5I/C59R/S108N/I164L. Residue N108 is the cause of pyrimethamine resistance with the highest repulsive interaction energy
modeling of 31 pyrimethamine derivatives into the active site of dihydrofolate reductase obtained from crystal structures 1J3I.pdb and 1J3K.pdb. Evaluation of predicted binding modes and key protein-ligand interactions
modeling of 32 pyrimethamine derivatives into the active site of dihydrofolate reductase obtained from crystal structure 1J3K.pdb. Evaluation of predicted binding modes and key protein-ligand interactions
-
modified microbatch method with PRG 4000 as the primary precipitating agent, crystals belong to the orthorhombic space group P2(1)2(1)2(1), with two molecules per asymmetric unit and 52% solvent content
-
wild-type and pyrimethamine-resistant mutant S58R/S117N in complex with NADPH and inhibitors pyrimethamine or 6-ethyl-5-phenylpyrimidine-2,4-diamine
crystallization of refolded recombinant enzyme as ternary complex with NADPH and various inhibitors
-
modeling data of inhibitor PT682 in the active site indicate that binding would require ligand-induced conformational changes to the enzyme for the inhibitor to fit or that the inhibitor side-chain would have to adopt an alternative binding mode to that observed for similar inhibitors. The complexes have an increased active-site volume compared with complexes of the enzyme from Mus musculus
-
purified enzyme complexed with inhibitor N6-methyl-N6-1-naphthylpyrido[2,3-d]pyrimidine-2,4,6-triamine, and NADPH, hanging drop vapor diffusion method, mixing of 11.4-14.1 mg/ml protein in 50 mM MES, pH 6.0, 100 ml KCl, and a tenfold excess of NADPH and inhibitor, with reservoir solution containiing 35% PEG 2000, 49 mM MES, pH 6.0, and 100 mM KCl, equilibration against a reservoir solution consisting of 0.1 M HEPES pH 7.5, 25% PEG 2000 MM, X-ray diffraction structure determination and analyis at 1.61 A resolution, modeling
purified recombinant detagged wild-type enzyme and mutant K37Q in complex with inhibitor 2,4-diamino-6-[(2',5'-dichloro anilino)methyl]pyrido[2,3-d]pyrimidine, hanging drop vapour diffusion method, mixing of 20.4 mg/ml protein in 20 mM MES, pH 6.0, 100 mM KCl with a 0:1 molar excess of NADPH and inhibitor, and with reservoir solution containing 100 mM K2HPO4, pH 6.9, 60-64% saturated (NH4)2SO4, and 3% (v/v) ethyl alcohol, 0.01 ml drops, 4°C, for mutant K37Q/F69N, 10.9 mg/ml protein in 10 mM MES, pH 6.0, and 100 mM KCl, is mixed with with a 0:1 molar excess of NADPH and inhibitor, and with a reservoir solution containing 33-36% PEG 2000, 50 mM MES, pH 6.0, and 100 mM KCl, 14°C, X-ray diffraction structure determination and analysis at 2.2 A resolution, modeling
purified enzyme complexed with inhibitor N6-methyl-N6-1-naphthylpyrido[2,3-d]pyrimidine-2,4,6-triamine, and NADPH, X-ray diffraction structure determination and analyis
purified recombinant His-tagged enzyme, mixing of 0.001 ml of 8 mg/ml protein solution with 0.001 ml of well solution containing 100 mM Tris-HCl, pH 8.0, and 2.0 M ammonium sulfate, at 18°C, two weeks, X-ray diffraction structure determination and analysis at 1.95 A resolution, molecular replacement using the modified structure of chicken liver DHFR, PDB ID 8DFR, as search model
crystal structure of the wild-type chromosomal DHFR from Staphylococcus aureus in complex with NADPH and trimethoprim is determined to 1.95 A resolution. The enzyme maintains the conserved fold of DHFR observed in other species with the active site core formed by an eight-stranded beta-sheet and four alpha-helices surrounding the core
purified recombinant wild-type and mutant enzymes bound to cofactor NADPH and inhibitors 5, 8, 10, and 15, hanging-drop vaporization method, 12 mg/ml protein is incubated with 1 mM inhibitor and 2 mM NADPH on ice for 2 h, mixing of equal volumes of the protein:ligand:NADPH solution with an optimized crystallization solution consisting of 15% PEG 10000, 150 mM sodium acetate, 100 mM MES, pH 6.5, and 5% butyrlactone, 5-7 days X-ray diffraction structure determination and analysis at 1.69-2.35 A resolutions, overview
purified recombinant wild-type and mutant enzymes in complex with inhibitor trimethoprim, hanging drop vapour diffusion method, 30.6 mg/ml wild-type enzyme protein mixed with 1 mM NADPH and 1 mM trimethoprim and incubated on ice for 3 h, mixing the protein 1:1 with a reservoir solution containing 30 mM citric acid/40 mM bis-tris propane pH 6.4, 13.3% PEG 3350, and 16.7% PEG 6000 and incubating at 22°C, hexagonal rod crystals form in 1-2 weeks, X-ray diffraction structure determination and analysis at 3.0 and 1.95 A resolution, respectively, molecular replacement and structure modelling
wild-type enzyme or mutant F98Y in ternary complex with cofactor NADPH and inhibitors AR-101, AR-102, or iclaprim, hanging-drop vapour-diffusion technique at 24°C, mixing of 30 mg/ml protein with 5 mM NADPH and 0.3 mg/ml inhibitor in 25% PEG 3350, 200 mM NaCl, and 100 mM bis-tris, pH 5.5, X-ray diffraction structure determination and analysis at 2.1-2.5 A resolution
vapour diffusion technique at 18°C, two high-resolution structures of dihydrofolate reductase in its unliganded state, and in its ternary complex with the cofactor NADPH and the inhibitor, methotrexate
purified recombinant wild-type TS-DHFR enzyme and truncated TS-DHFR mutant lacking the surface loops, complexed with dUMP and NADPH, as well as with inhibitors methotrexate and N10-propargyl-5,8-dideazafolate, 10 mg/ml protein with 10 mM of each ligand is mixed with mixed with 18% PEG 3350, 0.1 M potassium formate in a 1:1 ratio, 4-6 days, X-ray diffraction structure determination and analysis at 3.7 A and 2.2 A resolution, respectively
in complex with NADPH and inhibitors, hanging drop vapor diffusion method, using
-
purified recombinant His-tagged TbDHFR-TS, sitting drop vapour diffusion method, a few days at at 8°C or at room temperature, drops consist of 0.001 ml of protein solution and 0.001 ml of precipitant equilibrated against a 0.2 ml of reservoir solution. No crystal growth is observed over one year of incubation, preventing to obtain structural information on the enzyme
structure-based three-dimensional quantitative structure–activity relationship 3D-QSAR approach to predict the biochemical activity for inhibitors of Trypanosoma cruzi dihydrofolate reductase-thymidylate synthase. Crystal structures of complexes of the enzymewith eight different inhibitors of the DHFR activity together with the structure in the substrate-free state have been used to validate and refine docking poses of ligands
comparison of the backbone conformation in crystal structures, detection of mutational insertions
-
purified recombinant enzyme, hanging drop vapor diffusion method, 13 mg/ml protein in 20 mM Tris pH 8.0, 20% glycerol, 0.1 mM EDTA and 2 mM DTT, incubation with 1.5 mM NADPH, mixing with an equal volume of crystallization solution containing 0.1 M Tris, pH 8.5, 35-40% PEG 4000 and 0.3-0.4 M MgCl2, X-ray diffraction structure determination and analysis at 1.95-2.11 A resolution