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)

Substitution of glycine or alanine for phenylalanine 31 in human dihydrofolate reductase produces variants that are inhibited less by methotrexate (MTX) than the previously reported serine variant. The 100 times decrease in MTX affinity for the glycine variant is due to slower binding, and to inability of the initial complex to isomerize to a nondissociating conformer. A polar group at position 31 is unnecessary for resistance, but residues larger than serine confer no resistance. The glycine variant best fulfills criteria for gene therapy: low Km for H2folate, high kcat, and good stability. Although kcat is unaltered by these mutations, the rate of hydride transfer is greatly decreased. Presteady-state measurements have enabled a complete catalytic scheme to be constructed for the glycine variant that predicts observed steady-state behavior. The crystal structures of inhibitor complexes of the serine, alanine, and glycine mutants and of the wild-type enzyme show that the mutations cause little perturbation of the protein backbone, of side chains of residues at the active site, or of the bound inhibitor. A molecule of bound water occupies the space vacated by the phenyl group.
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PMID:Methotrexate-resistant variants of human dihydrofolate reductase. Effects of Phe31 substitutions. 814 41

These structural studies reveal unusual intermolecular interactions for the binding of inhibitors and cofactor in ternary complexes with both wild type and F31 mutant recombinant human DHFR and show that these inhibitors have flexibility in occupying the active site. These studies also possibly indicate the first structural data for a ternary complex with a folate inhibitor and a polyglutamate side chain. However, further refinement of this data is necessary before this can be confirmed. In contrast to the ternary complexes of folate and MTX, the lipophilic antifolate PTX binds with its methoxybenzoyl ring oriented toward the cofactor nicotinamide ring, while that of TMQ it is bound closer to the Phe-31 position. Furthermore, the nicotinamide ring makes a close contact to the N10 amine of TMQ, significantly different from its binding site interactions in MTX complexes. These data also reveal that the conserved contacts between the cofactor carboxyamide with the enzyme backbone residues Ala-9 and Ile-16 are dictated by the enzyme and that changes in the orientation of the structural elements requires only subtle changes in the secondary structural units in which they are contained. Therefore, only by careful analysis of a series of enzyme complexes can the mechanisms of binding action be delineated.
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PMID:Conformational analysis of human dihydrofolate reductase inhibitor complexes: crystal structure determination of wild type and F31 mutant binary and ternary inhibitor complexes. 830 63

The role of the 3'-carboxamide substituent of NADPH in the reduction of pteridine substrates as catalyzed by dihydrofolate reductase (EC 1.5.1.3, DHFR) has been investigated by determining crystal structures at 2.3 A of chicken liver DHFR in a binary complex with oxidized thionicotinamide adenine dinucleotide (thioNADP+) and in a ternary complex with thioNADP+ and biopterin. These structures are isomorphous with those previously reported for chicken liver DHFR [Volz, K.W., Matthews, D.A., Alden, R.A., Freer, S. T., Hansch, C., Kaufman, B. T., & Kraut, J. (1982) J. Biol. Chem. 257, 2528-2536]. ThioNADPH, which has a 3'-carbothioamide substituent in place of a 3'-carboxamide, functions very poorly as a coenzyme for DHFR [Williams, T. J., Lee, T. K., & Dunlap, R. B. (1977) Arch, Biochem. Biophys. 181, 569-579; Stone, S. R., Mark, A., & Morrison, J. F. (1984) Biochemistry 23, 4340-4346]. Comparisons show that, while NADP+ and NADPH bind to DHFR with the pyridine ring and 3'-carboxamide coplanar, the thioamide group is twisted by 23 degrees from the pyridine plane in both the binary and ternary complexes. This twist appears to be due to steric conflict between the thioamide sulfur atom and both the pyridine ring at C4 and the adjacent protein backbone at Ala-9. It results in an unfavorably close contact between the sulfur and the biopterin pteridine ring (0.9 A less than the van der Waals separation) which, on the basis of the refined structure, greatly destabilizes the binding of biopterin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Crystal structures of chicken liver dihydrofolate reductase: binary thioNADP+ and ternary thioNADP+.biopterin complexes. 833 18

The use of synthetic tRNA for in vitro protein engineering was tested in a coupled transcription/translation system prepared from Escherichia coli. DNA sequences similar to the natural tRNA(Ala/UGC) gene from E. coli but with different anticodons were synthesized in vitro, cloned into a DNA plasmid, and then transcribed in vitro with T7 RNA polymerase. The UGC alanine anticodon was changed to CUA corresponding to the UAG stop codon, CCU corresponding to the rarely used AGG arginine codon, and two four-nucleotide anticodons used to suppress stop codons. Bacterial dihydrofolate reductase was the test protein. Its cloned coding sequence was mutagenized at the GUG codon for valine-75 to correspond to the anticodons of the tRNA constructs, and then the plasmids were used to direct the synthesis of dihydrofolate reductase in the coupled transcription/translation system containing the corresponding synthetic tRNA. The results indicate that all four synthetic tRNAs were functionally active in the synthesis of full-length, enzymatically active dihydrofolate reductase protein.
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PMID:In vitro protein engineering using synthetic tRNA(Ala) with different anticodons. 834 99

The gene coding for the enzyme dihydrofolate reductase of the extremely halophilic archaebacterium Haloferax volcanii was recombined into the Escherichia coli expression vector pET11d. Following induction, the enzyme was produced in large quantities and accumulated in the cells in an insoluble form. The enzymic activity could be efficiently reconstituted by dissolving the aggregate in 6 M guanidine hydrochloride followed by dilution into salt solutions. Mutants were produced in which Lys30 was converted to Leu (K30L), Lys31 was converted to Ala (K31A) and a double mutant in which both lysines were converted (K30L, K31A). The mutated enzymes were produced in E. coli, activated and purified to homogeneity. The effect of the salt concentration on the steady-state kinetic parameters was determined. It was found that the salt concentration affects the Km but not kcat of the various mutants.
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PMID:High expression in Escherichia coli of the gene coding for dihydrofolate reductase of the extremely halophilic archaebacterium Haloferax volcanii. Reconstitution of the active enzyme and mutation studies. 836 6

As a result of the expression of a single open reading frame composed of the coding sequence for a cysteine-free mutant (Cys85-->Ala, Cys152-->Ser) of Escherichia coli dihydrofolate reductase (DHFR; 18K monomeric protein), that for the E. coli thymidylate synthase (TS; dimeric protein with a 30K promoter), and a spacer sequence (coding 7 amino acids) with a Shine-Dargarno sequence, an active hetero-dimeric bifunctional enzyme with 50K DHFR-TS and 30K TS polypeptides was stably produced in the transformed E. coli cell in addition to an overproduction of the TS dimeric enzyme. The highly purified hetero-dimeric enzyme has similar Vmax and Km values in both DHFR and TS activities to those of the natural counterparts, monomeric DHFR and dimeric TS. Although the hetero-dimeric enzyme did not show an apparent channeling transfer of dihydrofolate (the intermediate substrate) between the spatially discrete DHFR and TS active sites, the coupling efficiency of the TS and DHFR reactions in the artificial enzyme was better than that in the separated enzymes, as shown by a decrease in the intermediate concentration at the steady state in the coupled reaction.
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PMID:Production of hetero-dimeric dihydrofolate reductase-thymidylate synthase bifunctional enzyme. 853 27

Despite several similarities in structure and kinetic behavior, the bacterial and vertebrate forms of the enzyme dihydrofolate reductase (DHFR) exhibit differential specificity for folate. In particular, avian DHFR is 400 times more specific for folate than the Escherichia coli reductase. We proposed to enhance the specificity of the E. coli reductase for folate by incorporating discrete elements of vertebrate secondary structure. Two vertebrate loop mutants, VLI and VLII containing 3-7 additional amino acid insertions, were constructed and characterized by using steady-state kinetics, spectrofluorimetric determination of ligand equilibrium dissociation constants, and circular dichroism spectroscopy. Remarkably, the VLI and VLII mutants are kinetically similar to wild-type E. coli reductase when dihydrofolate is the substrate, although VLII exhibits prolonged kinetic hysteresis. Moreover, the VLI dihydrofolate reductase is the first mutant form of E. coli DHFR to display enhanced specificity for folate [(kcat/Km)mutant/(kcat/Km)wt = 13]. A glycine-alanine loop (GAL) mutant was also constructed to test the design principles for the VLI mutant. In this mutant of the VLI reductase, all of the residues from positions 50 to 60, except the strictly conserved amino acids Leu-57 and Arg-60, were converted to either glycine or alanine. A detailed kinetic comparison of the GAL and wild-type reductases revealed that the mutations weaken the binding by both cofactor and substrate by up to 20-fold, but under saturating conditions the enzyme exhibits a kcat value nearly identical to that of the wild type. The rate of hydride transfer is reduced by a factor of 30, with a compensating increase in the dissociation rate for tetrahydrofolate. Although key stabilizing interactions have been sacrificed (it shows no activity toward folate), the maintenance of the correct register between key residues preserves the activity of the enzyme toward its natural substrate. Collectively, neither specific proximal point site mutations nor larger, more distal secondary structural substitutions are sufficient to confer a specificity for folate reduction that matches that observed with the avian enzyme. This is consistent with the hypothesis that the entire protein structure must contribute extensively to the enzyme's specificity.
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PMID:Engineering specificity for folate into dihydrofolate reductase from Escherichia coli. 863 97

To elucidate the role of a flexible loop (residues 64-72) in the stability and function of Escherichia coli dihydrofolate reductase, glycine-67 in this loop was substituted by site-directed mutagenesis with seven amino acids (Ala, Cys, Asp, Leu, Ser, Thr, and Val). The circular dichroism spectra suggested that the confirmation of the native structure was affected by the mutations in both the presence and absence of NADPH. The free energy change of unfolding by urea decreased in the order of G67A > G67S > or = wild-type > or = G67D > G67T > G67C > or = G67L > G67V. The steady-state kinetic parameters for the enzyme reaction, Km and kcat, were only slightly influenced, but the rate of the hydride transfer reaction was significantly changed by the mutations, as revealed by the deuterium isotope effect on the enzyme activity. These results suggest that site 67 in the flexible loop, being very far from the active site, plays an important role in the stability and function of this enzyme. The characteristics of the mutations were discussed in terms of the modified flexibility of the native structure, compared with the results of mutations at site 121 in another flexible loop.
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PMID:Effects of point mutations at the flexible loop glycine-67 of Escherichia coli dihydrofolate reductase on its stability and function. 874 72

Short peptides which contained a single Cys residue were introduced into both N- and C-termini of the Cys-free mutant of DHFR (Cys85 --> Ala, Cys152 --> Ser double mutant) by a recombinant DNA method, then the terminal regions were connected through a disulfide bond by oxidation. The oxidized form and reduced form proteins have as high enzymatic activity as wild-type DHFR. There is no detectable difference between the CD spectra of the reduced and oxidized forms at low (15 degrees C, native condition) and high temperature (80 degrees C, unfolded condition). The thermal transition of the oxidized proteins at the concentration of 0.15 mg/ml (8.5 microM) is completely reversible as demonstrated by the CD spectra. No aggregated materials were detected in the oxidized protein on gel-filtration HPLC after heat treatment up to the protein concentration of 0.5 mg/ml. The reduced protein, however, even in the presence of reducing agent, showed only partial reversibility, with as much as 55 and 95% of the heat-treated protein at the concentrations of 0.15 and 0.5 mg/ml being eluted as the high molecular aggregated form, respectively. The apparent transition temperatures (Tm) of the oxidized forms were 5-7 degrees C higher than those of the reduced counterparts. The oxidized protein that had been denatured with guanidine-HCl was eluted later than the denatured reduced protein on gel-filtration HPLC in the presence of 5 M guanidine-HCl. The limitation of spatial movement of the termini may prevent intermolecular interaction of exposed domains during denaturation-renaturation process, giving rise to the irreversible denaturation. The flexibility of the terminal is also suggested to be an important factor for improving thermal stability of proteins.
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PMID:Stability and reversibility of thermal denaturation are greatly improved by limiting terminal flexibility of Escherichia coli dihydrofolate reductase. 883 33

The effects of six amino acid substitutions in Lactobacillus casei dihydrofolate reductase, predominantly in the coenzyme binding site, on catalysis and on the negative cooperativity between NADPH and tetrahydrofolate binding have been determined. Replacement of Leu62, His64 or Leu54 by alanine has no effect on kcat, and produces only modest changes in negative cooperativity. Alanine substitution of His77, which interacts indirectly with the coenzyme adenine ring, leads to a doubling of the negative cooperativity and a consequent doubling of kcat. Replacement of Arg43, which interacts with the coenzyme 2'-phosphate, by alanine, or of Trp21, which interacts with the coenzyme nicotinamide ring, by histidine leads to a 20-100-fold decrease in negative cooperativity. In both mutants there is a decrease in kcat; isotope effects show that product release is largely rate-limiting in R43A, whereas in W21H hydride ion transfer is rate-limiting. 1H NMR has been used to obtain information on the extent of the structural changes produced by the substitutions. This varies from very local effects in H64A to very widespread effects in W21H. These changes are used as the basis for discussion of the mechanisms of the functional effects of the various substitutions. It is suggested that residues in helix C, beta-strand 3 and the beta3-beta4 loop may be involved in the transmission of effects between the coenzyme and substrate binding sites.
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PMID:Effects of single-residue substitutions on negative cooperativity in ligand binding to dihydrofolate reductase. 934 47


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