Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.5.1.3 (dihydrofolate reductase)
5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Streptococcus pneumoniae isolates resistant to several antimicrobial agent classes including trimethoprim-sulfamethoxazole have been reported with increasing frequency throughout the world. The MICs of trimethoprim, sulfamethoxazole, and trimethoprim-sulfamethoxazole (1:19) for 259 clinical isolates from South Africa were determined, and 166 of these 259 (64%) isolates were resistant to trimethoprim-sulfamethoxazole (MICs > or =20 mg/liter). Trimethoprim resistance was found to be more strongly correlated with trimethoprim-sulfamethoxazole resistance (correlation coefficient, 0.744) than was sulfamethoxazole resistance (correlation coefficient, 0.441). The dihydrofolate reductase genes from 11 trimethoprim-resistant (MICs, 64 to 512 microg/ml) clinical isolates of Streptococcus pneumoniae were amplified by PCR, and the nucleotide sequences were determined. Two main groups of mutations to the dihydrofolate reductase gene were found. Both groups shared six amino acid changes (Glu20-Asp, Pro70-Ser, Gln81-His, Asp92-Ala, Ile100-Leu, and Leu135-Phe). The first group included two extra changes (Lys60-Gln and Pro111-Ser), and the second group was characterized by six additional amino acid changes (Glu14-Asp, Ile74-Leu, Gln91-His, Glu94-Asp, Phe147-Ser, and Ala149-Thr). Chromosomal DNA from resistant isolates and cloned PCR products of the genes encoding resistant dihydrofolate reductases were capable of transforming a susceptible strain of S. pneumoniae to trimethoprim resistance. The inhibitor profiles of recombinant dihydrofolate reductase from resistant and susceptible isolates revealed that the dihydrofolate reductase from trimethoprim-resistant isolates was 50-fold more resistant (50% inhibitory doses [ID50s], 3.9 to 7.3 microM) than that from susceptible strains (ID50s, 0.15 microM). Site-directed mutagenesis experiments revealed that one mutation, Ile100-Leu, resulted in a 50-fold increase in the ID50 of trimethoprim. The resistant dihydrofolate reductases were characterized by highly conserved redundant changes in the nucleotide sequence, suggesting that the genes encoding resistant dihydrofolate reductases may have evolved as a result of inter- or intraspecies recombination by transformation.
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PMID:Mutations in the dihydrofolate reductase gene of trimethoprim-resistant isolates of Streptococcus pneumoniae. 937 41

Two-dimensional heteronuclear (1H-15N) nuclear magnetic relaxation studies of dihydrofolate reductase (DHFR) from Escherichia coli have demonstrated that glycine-121 which is 19 A from the catalytic center of the enzyme has large-amplitude backbone motions on the nanosecond time scale [Epstein, D. M., Benkovic, S. J., and Wright, P. E. (1995) Biochemistry 34, 11037-11048]. In order to probe the dynamic-function relationships of this residue, we constructed a mutant enzyme in which this glycine was changed to valine. Equilibrium binding studies indicated that the Val-121 mutant retained wild-type binding properties with respect to dihydrofolate and tetrahydrofolate; however, binding to NADPH and NADP+ was decreased by 40-fold and 2-fold, respectively, relative to wild-type DHFR. Single-turnover experiments indicated that hydride transfer was reduced by 200-fold to a rate of 1.3 s-1 and was the rate-limiting step in the steady state. Interestingly, pre-steady-state kinetic analysis of the Val-121 mutant revealed a conformational change which preceded chemistry that occurred at a rate of 3.5 s-1. If this step exists in the kinetic mechanism of the wild-type enzyme, then it would be predicted to occur at a rate of approximately 2000 s-1. Glycine-121 was also changed to alanine, serine, leucine, and proline. While the Ala-121 and Ser-121 mutants behaved similar to wild-type DHFR, the Leu-121 and Pro-121 mutants behaved like Val-121 DHFR in that hydride transfer was the rate-limiting step in the steady state and a conformational change preceding chemistry was observed. Finally, insertion of a glycine or valine between amino acids 121 and 122 produced mutant enzymes with properties similar to wild-type or Val-121 DHFRs, respectively. Taken together, these results provide compelling evidence for dynamic coupling of a remote residue to kinetic events at the active site of DHFR.
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PMID:Evidence for a functional role of the dynamics of glycine-121 of Escherichia coli dihydrofolate reductase obtained from kinetic analysis of a site-directed mutant. 939 9

To elucidate the role of a flexible loop (residues 142-149) in the stability and function of Escherichia coli dihydrofolate reductase, alanine-145 in this loop was substituted by site-directed mutagenesis with ten amino acids (Glu, Phe, Gly, His, Ile, Leu, Arg, Ser, Thr, and Val). The amount of three mutant proteins (A145E, A145I, and A145L) in cells was too small to allow the measurement of circular dichroism (CD) spectra and urea unfolding. The CD spectra of other seven mutants were identical with those of the wild-type DHFR, indicating that the native conformation of DHFR was not affected by the mutations. The free energy change of unfolding by urea decreased with an increase in the hydrophobicity of amino acid residues introduced, A145T>A145R>A145G>=A145S>=A145H>A145V++ +>wild-type>=A145F. The steady-state kinetic parameters for the enzyme reaction, Km and ksub, were only slightly influenced by the mutations. These results suggest that site 145 in the flexible loop plays an important role in the stability but has little or no effect on the native structure and function of this enzyme. The characteristics of the mutations are discussed in comparison with those of mutations at site 67 [Ohmae et al. (1996) J. Biochem. 119, 703-710] and at site 121 [Gekko et al. (1994) J. Biochem. 116, 34-41] in two other flexible loops.
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PMID:Effects of point mutations at the flexible loop alanine-145 of Escherichia coli dihydrofolate reductase on its stability and function. 956 14

On the basis of X-ray crystallographic data, Sawaya and Kraut proposed that Met20 loop conformational changes modulate ligand specificity observed in the catalytic cycle for Escherichia coli dihydrofolate reductase (DHFR) [Sawaya, M. R., and Kraut, J. (1997) Biochemistry 36, 586-603]. Interloop hydrogen bonds stabilize either a closed Met20 loop conformation observed in substrate complexes or an occluded Met20 loop conformation observed in product complexes, respectively. To test this model, we targeted a single hydrogen bond occurring exclusively in the closed Met20 loop conformation. Specifically, Asp122 in the betaF-betaG loop was independently substituted with asparagine, serine, and alanine-amino acids with decreasing abilities to hydrogen-bond. The kinetic analyses of the Asp122 mutants enabled the construction of kinetic schemes at pH 7.0 that demonstrate two striking features. First, a significant correlation exists between decreased binding of nicotinamide adenine dinucleotide phosphate, reduced (NADPH), and decreased hydride transfer rates resulting from these mutations. In other words, the interactions of Asp122 are along the reaction coordinate leading to the transition state. Second, substitutions for Asp122 alter the catalytic pathway preferred by wild-type DHFR under saturating conditions of substrate and cofactor. Overall, the steady-state rate contains contributions from the product off rates from the DHFR.5,6, 7,8-tetrahydrofolate (H4F) and DHFR.NADPH.H4F complexes and from the rate of hydride transfer. These mutational effects support the mechanistic model whereby interloop contacts regulate an equilibrium of Met20 loop conformations that, in turn, modulate ligand affinity and turnover.
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PMID:Strength of an interloop hydrogen bond determines the kinetic pathway in catalysis by Escherichia coli dihydrofolate reductase. 957 48

The efficacy of sulphadoxine/pyrimethamine (S/P) in treatment of uncomplicated falciparum malaria in Africa is increasingly compromised by development of resistance. The occurrence of mutations associated with the active site sequence in the Plasmodium falciparum genes coding for dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) is associated with in vitro resistance to pyrimethamine and sulphadoxine. This study investigates the occurrence of these mutations in infected blood samples taken from Tanzanian children before treatment with S/P and their relationship to parasite breakthrough by day 7. The results show that alleles of DHPS (436-alanine, 437-alanine and 540-lysine) were significantly reduced in prevalence on day 7 after S/P treatment. In this area, a DHPS with 436-serine, 437-glycine and 540-glutamate appears to play a major role in resistance to S/P in vivo. Evidence for the influence of mutations in the DHFR gene in this investigation is not clear, probably because of the high prevalence of 'resistance-related' mutations at day 0 in the local parasite population. For apparently the same reason, it was not possible to show a statistical association between S/P resistance and the presence of particular polymorphisms in the DHFR and DHPS genes before treatment.
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PMID:Polymorphisms in the dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) genes of Plasmodium falciparum and in vivo resistance to sulphadoxine/pyrimethamine in isolates from Tanzania. 973 30

The E2F family of heterodimeric transcription factors plays an important role in the regulation of gene expression at the G1/S phase transition of the mammalian cell cycle. Previously, we have demonstrated that cell cycle regulation of murine dihydrofolate reductase (dhfr) expression requires E2F-mediated activation of the dhfr promoter in S phase. To investigate the mechanism by which E2F activates an authentic E2F-regulated promoter, we precisely replaced the E2F binding site in the dhfr promoter with a Gal4 binding site. Using Gal4-E2F1 derivatives, we found that E2F1 amino acids 409-437 contain a potent core transactivation domain. Functional analysis of the E2F1 core domain demonstrated that replacement of phenylalanine residues 413, 425, and 429 with alanine reduces both transcriptional activation of the dhfr promoter and protein-protein interactions with CBP, transcription factor (TF) IIH, and TATA-binding protein (TBP). However, additional amino acid substitutions for phenylalanine 429 demonstrated a strong correlation between activation of the dhfr promoter and binding of CBP, but not TFIIH or TBP. Finally, transactivator bypass experiments indicated that direct recruitment of CBP is sufficient for activation of the dhfr promoter. Therefore, we suggest that recruitment of CBP is one mechanism by which E2F activates the dhfr promoter.
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PMID:Activation of the murine dihydrofolate reductase promoter by E2F1. A requirement for CBP recruitment. 1033 93

In vivo testing for resistance of Plasmodium falciparum to co-trimoxazole (trimethoprim/sulfamethoxazole) was performed in Uganda in 41 children with uncomplicated malaria, and blood samples were screened before and after treatment for polymorphisms in the antifolate target genes for dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS). Selection towards a specific genotype at some codons of the DHFR and DHPS genes was observed in samples collected after exposure to co-trimoxazole drug pressure. The alleles 51-isoleucine, 59-arginine, and 108-serine of DHFR were significantly associated with clinical resistance, as was allele 581-alanine of DHPS. Resistance against antifolate combinations probably requires resistance-related polymorphisms in both the DHFR and the DHPS genes. In addition, it appears that the trimethoprim-resistant DHFR genotype differs from that for pyrimethamine at residue 108.
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PMID:Plasmodium falciparum: selection of serine 108 of dihydrofolate reductase during treatment of uncomplicated malaria with co-trimoxazole in Ugandan children. 1043 69

The efficacy of sulfadoxine/pyrimethamine (S/P) in treatment of uncomplicated falciparum malaria in Africa is increasingly compromised by development of resistance. The occurrence of active site mutations in the Plasmodium falciparum gene sequences coding for dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) is known to confer resistance to pyrimethamine and sulfadoxine. This study investigated the occurrence of these mutations in infected blood samples taken from Ugandan children before treatment with S/P and their relationship to parasite breakthrough by day 7. The results confirm the occurrence of mutations in DHFR and DHPS that were significantly selected under S/P pressure at day 7: a combination of alleles 51-isoleucine and 108-asparagine in DHFR, and 436-serine, 437-alanine, 540-lysine and 581-alanine in DHPS, appears to play a major role in the development of in vivo resistance in P. falciparum strains against S/P. Therefore, earlier results derived from isolates from hyperendemic areas in Tanzania were confirmed by this investigation.
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PMID:Plasmodium falciparum resistance to sulfadoxine/pyrimethamine in Uganda: correlation with polymorphisms in the dihydrofolate reductase and dihydropteroate synthetase genes. 1049 91

Molecular docking programs screen chemical databases for novel ligands that fit protein binding sites. When one compound fits the site well, close analogs typically do the same. Therefore, many of the compounds that are found in such screens resemble one another. This reduces the variety and novelty of the compounds suggested. In an attempt to increase the diversity of docking hit lists, the Available Chemicals Directory was grouped into families of related structures. All members of every family were docked and scored, but only the best scoring molecule of a high-ranking family was allowed in the hit list. The identity and scores of the other members of these families were recorded as annotations to the best family member, but they were not independently ranked. This family-based docking method was compared with molecule-by-molecule docking in screens against the structures of thymidylate synthase, dihydrofolate reductase (DHFR), and the cavity site of the mutant T4 lysozyme Leu99 --> Ala (L99A). In each case, the diversity of the hit list increased, and more families of known ligands were found. To investigate whether the newly identified hits were sensible, we tested representative examples experimentally for binding to L99A and DHFR. Of the six compounds tested against L99A, five bound to the internal cavity. Of the seven compounds tested against DHFR, six inhibited the enzyme with apparent K(i) values between 0.26 and 100 microM. The segregation of potential ligands into families of related molecules is a simple technique to increase the diversity of candidates suggested by database screens. The general approach should be applicable to most docking methods. Proteins 2001;42:279-293.
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PMID:Docking molecules by families to increase the diversity of hits in database screens: computational strategy and experimental evaluation. 1111 52

As a continuation to our studies on the importance of interloop interactions in the Escherichia coli DHFR catalytic cycle, we have investigated the role of the betaG-betaH loop in modulating the closed and occluded conformations of the Met20 loop during the DHFR catalytic cycle. Specifically, to assess the importance of the hydrogen bond formed between Ser148 in the betaG-betaH loop and the Met20 loop, Ser148 was independently substituted with aspartic acid, alanine, and lysine. Moreover, the betaG-betaH loop was deleted entirely to yield the Delta(146-148) DHFR mutant. Steady-state turnover rates for all mutants were at most 3-fold lower than the wild-type rate. Lack of an isotope effect on this rate indicated the chemistry step does not contribute to the steady-state turnover. Consistent with this finding, hydride transfer rates for the DHFR mutants were at least 10-fold greater than the observed steady-state rates. The values ranged from a 30% decrease (Ser148Ala and Ser148Lys) to a 50% increase (Ser148Asp) in rate relative to that of the wild type. Modifications of the betaG-betaH loop enhanced the affinity for the cofactor and decreased the affinity for pterin, as determined by the K(D) values of the mutant proteins. Further analysis of Ser148Ala and Delta(146-148) DHFRs indicated these effects were manifest mainly in ligand off rates, although in some cases the on rate was affected. The Ser148Asp and Delta(146-148) mutations perturbed the preferred catalytic cycle through the introduction of branching at key intermediates. Rather than following the single WT pathway which involves loss of NADP(+) and rebinding of NADPH to precede loss of the product H4F (negative cooperativity), the mutants can reenter the catalytic cycle through different pathways. These findings suggest that the role of the interloop interaction between the betaG-betaH loop and the Met20 loop is to modulate ligand off rates allowing for proper cycling through the preferred kinetic pathway.
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PMID:Interloop contacts modulate ligand cycling during catalysis by Escherichia coli dihydrofolate reductase. 1117 Apr 7


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