Purification and characterization of selenomethionyl thymidylate synthase from Escherichia coli: comparison with the wild-type enzyme

Replacement of methionine (Met) residues by selenomethionine (SeMet) was recently shown to facilitate the crystallographic analysis of protein structure through the application of multi-wavelength anomalous diffraction techniques [Yang et al. (1990) Science (Washington, D.C.) 249, 1398-1405]. The availability of SeMet-containing proteins provides an excellent opportunity to evaluate the effects of the complete replacement of Met by SeMet. We chose to compare the properties of selenomethionyl thymidylate synthase isolated from Escherichia coli DL41 (a methionine auxotroph) and wild-type (wt) enzyme obtained from E. coli Rue10. An improved purification procedure for thymidylate synthase was developed which permitted the isolation of 25 mg of pure protein from 2 g of E. coli in 90% yield in no more than 8 h. The pure wt and SeMet enzymes exhibited specific activities 40% higher than published values. Thermal stability studies at 30 degrees C in degassed buffer showed that the SeMet enzyme (t1/2 67 h) was 8-fold less stable than wt enzyme (t1/2 557 h). The half-lives for the latter enzymes in nondegassed buffers at 30 degrees C were decreased by 2-fold, thus indicating the sensitivity of the enzyme to dissolved oxygen. Both enzymes exhibited essentially the same kinetic and binding properties, including Km(dUMP) (1.2 x 10(-6) M), specificity constant (1.6 x 10(6) s-1 M-1), and Kd for 5-fluorodeoxyuridylate binding (1.2 nM) in covalent inhibitory ternary complexes. In addition, X-ray crystallographic analysis by difference Fourier synthesis showed there was no significant difference in conformation between the SeMet enzyme and the wt enzyme.     

Dihydrofolate reductase. The stereochemistry of inhibitor selectivity

X-ray structural results are reported for 10 triazine and pyrimidine inhibitors of dihydrofolate reductase, each one studied as a ternary complex with NADPH and chicken dihydrofolate reductase. Analysis of these data and comparison with structural results from the preceding paper (Matthews, D.A., Bolin, J.T., Burridge, J.M., Filman, D.J., Volz, K.W., Kaufman, B. T., Beddell, C.R., Champness, J.N., Stammers, D.K., and Kraut, J. (1985) J. Biol. Chem. 260, 381-391) in which we contrasted binding of the antibiotic trimethoprim (TMP) to chicken dihydrofolate reductase on the one hand with its binding to Escherichia coli dihydrofolate reductase on the other, permit identification of differences that are important in accounting for TMP's selectivity. The crystallographic evidence strongly suggests that loss of a potential hydrogen bond between the 4-amino group of TMP and the backbone carbonyl of Val-115 when TMP binds to chicken dihydrofolate reductase but not when it binds to the E. coli reductase is the major factor responsible for this drug's more potent inhibition of bacterial dihydrofolate reductase. A key finding of the current study which is important in understanding why TMP binds differently to chicken and E. coli dihydrofolate reductases is that residues on opposite sides of the active-site cleft in chicken dihydrofolate reductase are about 1.5-2.0 A further apart than are structurally equivalent residues in the E. coli enzyme.     

Two distinct types of trimethoprim-resistant dihydrofolate reductase specified by R-plasmids of different compatibility groups.

R-Plasmids from a number of trimethoprim-resistant Escherichia coli and Citrobacter sp. were studied after transfer to E. coli K12 hosts. Each was found to specify a dihydrofolate reductase which was resistant to trimethoprim and Methotrexate, and which could be completely separated from the host chromosomal enzyme by gel filtration. Two distinct types of R-plasmid dihydrofolate reductases were identified. Type I enzymes, typified by the R483 enzyme previously described (Sk?ld, O., and Widh, A. (1974) J. Biol. Chem. 249, 4324-4325), are synthesized in amounts severalfold higher than the chromosomal enzyme. The 50% inhibitory concentrations (I50) of trimethoprim, Methotrexate, and aminopterin are increased several thousandfold over the corresponding values for the chromosomal enzyme. Type II R-plasmid dihydrofolate reductases are synthesized in about the same amount, or less, as the chromosomal enzyme, but are practically several hundredfold higher than those for the type I enzymes. Both types of R-plasmid dihydrofolate reductase showed little difference from the chromosomal enzyme in the binding of dihydrofolate, NADPH, folic acid, and 2,4-diaminopyrimidine.     

Homology of Escherichia coli B glutathione synthetase with dihydrofolate reductase in amino acid sequence and substrate binding site

Glutathione synthetase from Escherichia coli B showed amino acid sequence homology with mammalian and bacterial dihydrofolate reductases over 40 residues, although these two enzymes are different in their reaction mechanisms and ligand requirements. The effects of ligands of dihydrofolate reductase on the reaction of E. coli B glutathione synthetase were examined to find resemblances in catalytic function to dihydrofolate reductase. The E. coli B enzyme was potently inhibited by 7,8-dihydrofolate, methotrexate, and trimethoprim. Methotrexate was studied in detail and proved to bind to an ATP binding site of the E. coli B enzyme with K1 value of 0.1 mM. The homologous portion of the amino acid sequence in dihydrofolate reductases, which corresponds to the portion coded by exon 3 of mammalian dihydrofolate reductase genes, provided a binding site of the adenosine diphosphate moiety of NADPH in the crystal structure of dihydrofolate reductase. These analyses would indicate that the homologous portion of the amino acid sequence of the E. coli B enzyme provides the ATP binding site. This report gives experimental evidence that amino acid sequences related by sequence homology conserve functional similarity even in enzymes which differ in their catalytic mechanisms.     

Substrate-induced hysteresis in the activity of Escherichia coli dihydrofolate reductase

Full time course studies of the kinetic activity of Escherichia coli dihydrofolate reductase show that there is an increase in activity with time. The half-time for this hysteretic behavior is about 9 s. Preincubation of the enzyme with either of the substrates abolishes the lag and results in initial velocities which are 2-2.3-fold faster than those observed for the non-preincubated enzyme. The kinetic properties of the activated and nonactivated forms of the enzyme appear to be similar as measured by the full time course of the reaction. The results are consistent with observations for NADPH binding studies that the enzyme exists in two interconvertible forms, one of which is incapable of binding NADPH (Cayley, P. J., Dunn, S. M. J., and King, R. W. (1981) Biochemistry 20, 874-879).     

Characterization of Candida albicans dihydrofolate reductase

Dihydrofolate reductase from Candida albicans was purified 31,000-fold and characterized. In addition, the C. albicans dihydrofolate reductase gene was cloned into a plasmid vector and expressed in Escherichia coli, and the enzyme was purified from this source. Both preparations showed a single protein-staining band with a molecular weight of about 25,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzymes were stable and had an isoelectric point of pH 7.1 on gel isoelectric focusing. Kinetic characterization showed that the enzymes from each source had similar turnover numbers (about 11,000 min-1) and Km values for NADPH and dihydrofolate of 3-4 microM. Like other eukaryotic dihydrofolate reductases, the C. albicans enzyme exhibited weak binding affinity for the antibacterial agent trimethoprim (Ki = 4 microM), but further characterization showed that the inhibitor binding profile of the yeast and mammalian enzymes differed. Methotrexate was a tight binding inhibitor of human but not C. albicans dihydrofolate reductase; the latter had a relatively high methotrexate Ki of 150 pM. The yeast and vertebrate enzymes also differed in their interactions with KCl and urea. These two agents activate vertebrate dihydrofolate reductases but inhibited the C. albicans enzyme. The sequence of the first 36 amino-terminal amino acids of the yeast enzyme was also determined. This portion of the C. albicans enzyme was more similar to human than to E. coli dihydrofolate reductases (50% and 30% identity, respectively). Some key amino acid residues in the C. albicans sequence, such as E-30 (human enzyme numbering), were "vertebrate-like" whereas others, such as I-31, were not. These results indicate that there are physical and kinetic differences between the eukaryotic mammalian and yeast enzymes.     

Characterization of an R-plasmid dihydrofolate reductase with a monomeric structure

A plasmid-encoded dihydrofolate reductase that originated in a clinical isolate of Salmonella typhimurium (phage type 179) moderately resistant to trimethoprim has been isolated and characterized. The dihydrofolate reductase (called type III) was purified to homogeneity using a combination of gel filtration, hydrophobic chromatography, and methotrexate affinity chromatography. Polyacrylamide gel electrophoresis under denaturing and nondenaturing conditions indicated that the enzyme is a 16,900 molecular weight monomeric protein. Kinetic analyses showed that trimethoprim is a relatively tight binding inhibitor (Ki = 19 nM) competitive with dihydrofolate. The enzyme is also extremely sensitive to methotrexate inhibition (Ki = 9 pM) and has a high affinity for dihydrofolate (Km = 0.4 microM). The sequence of the first 20 NH2-terminal residues of the protein shows 50% homology with the trimethoprim-sensitive chromosomal Escherichia coli dihydrofolate reductase and suggests that the two enzymes may be closely related. This is the first example of a plasmid encoding for a monomeric dihydrofolate reductase only moderately resistant to trimethoprim, and a resistance mechanism, dependent in part on the high dihydrofolate affinity of the type III enzyme, is proposed.     

Porcine liver dihydrofolate reductase. Purification, properties, and amino acid sequence.

Porcine liver dihydrofolate reductase has been purified 18,000-fold to homogeneity. The properties of the purified enzyme were compared to those of dihydrofolate reductase from L1210 cells, the only mammalian reductase for which complete amino acid sequence data are available. The enzymes are very similar when compared on the basis of mechanism and kinetic constants, molecular weights, isoelectric points, and stimulation by salt. A comparison of the amino acid sequences of both enzymes shows an overall identity of 89%. Thus, the similarities seen in inhibitor-binding profiles of mammalian enzymes reflect the close relationship of these enzymes at the molecular level.     

Dihydrofolate reductase from amethopterin-resistant Lactobacillus casei. Sequences of the cyanogen bromide peptides and complete sequences of the enzyme.

The complete amino acid sequence of dihydrofolate reductase from an amethopterin-resistant strain of Lactobacillus casei has been determined by sequence analysis of peptides produced by cleavage with cyanogen bromide, trypsin, staphylococcal protease, and myxobacter protease. Comparison of this sequence with those of reductases from other bacterial sources shows that the enzymes are homologous. The Lactobacillus casei reductase sequences shows a 29% sequence identity with that of the Escherichia coli enzyme and a 34% identity with the sequence of the enzyme from Streptococcus faecium. The NH2-terminal 68 residues of the L. casei reductase show a 54% sequence identity with that of the enzyme from S. faecium.     

Characterization of the cloned Escherichia coli dihydrofolate reductase

Dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) was purified from Escherichia coli strains that carried derivatives of the multicopy recombinant plasmid, pJFM8. The results of enzyme kinetic and two-dimensional gel electrophoresis experiments showed that the cloned enzyme is indistinguishable from the chromosomal enzyme. Therefore it can be concluded that these strains are ideal for use as a source of enzyme for further studies on the biochemistry and regulation of this important enzyme. The plasmid derivatives were constructed by recloning experiments that utilized several restriction endonucleases. From the analysis both of these plasmids and the purified dihydrofolate reductase enzymes it was possible to deduce the location and orientation of the dihydrofolate reductase structural gene on the parent plasmid, pJFM8.     

Functional role of a mobile loop of Escherichia coli dihydrofolate reductase in transition-state stabilization.

The function of a highly mobile loop in Escherichia coli dihydrofolate reductase was studied by constructing a mutant (DL1) using cassette mutagenesis that had four residues deleted in the middle section of the loop (Met16-Ala19) and a glycine inserted to seal the gap. This part of the loop involves residues 16-20 and is disordered in the X-ray crystal structures of the apoprotein and the NADP+ binary complex but forms a hairpin turn that folds over the nicotinamide moiety of NADP+ and the pteridine moiety of folate in the ternary complex [Bystroff, C., & Kraut, J. (1991) Biochemistry 30, 2227-2239]. The steady-state and pre-steady-state kinetics and two-dimensional 1H NMR spectra were analyzed and compared to the wild-type protein. The kinetics on the DL1 mutant enzyme show that the KM value for NADPH (5.3 microM), the KM for dihydrofolate (2 microM), the rate constant for the release of the product tetrahydrofolate (10.3 s-1), and the intrinsic pKa value (6.2) are similar to those exhibited by the wild-type enzyme. However, the hydride-transfer rate declines markedly from the wild-type value of 950 s-1 to 1.7 s-1 for the DL1 mutant and when taken with data for substrate binding indicates that the loop contributes to substrate flux by a factor of 3.5 x 10(4). Thus, the mobility of loop I may provide a mechanism of recruiting hydrophobic residues which can properly align the nicotinamide and pteridine rings for the hydride-transfer process (a form of transition-state stabilization).(ABSTRACT TRUNCATED AT 250 WORDS)     

Catalytic activity of selenomethionine in removing amino acid, peptide, and protein hydroperoxides

Selenium is a critical trace element, with deficiency associated with numerous diseases including cardiovascular disease, diabetes, and cancer. Selenomethionine (SeMet; a selenium analogue of the amino acid methionine, Met) is a major form of organic selenium and an important dietary source of selenium for selenoprotein synthesis in vivo. As selenium compounds can be readily oxidized and reduced, and selenocysteine residues play a critical role in the catalytic activity of the key protective enzymes glutathione peroxidase and thioredoxin reductase, we investigated the ability of SeMet (and its sulfur analogue, Met) to scavenge hydroperoxides present on amino acids, peptides, and proteins, which are key intermediates in protein oxidation. We show that SeMet, but not Met, can remove these species both stoichiometrically and catalytically in the presence of glutathione (GSH) or a thioredoxin reductase (TrxR)/thioredoxin (Trx)/NADPH system. Reaction of the hydroperoxide with SeMet results in selenoxide formation as detected by HPLC. Recycling of the selenoxide back to SeMet occurs rapidly with GSH, TrxR/NADPH, or a complete TrxR/Trx/NADPH reducing system, with this resulting in an enhanced rate of peroxide removal. In the complete TrxR/Trx/NADPH system loss of peroxide is essentially stoichiometric with NADPH consumption, indicative of a highly efficient system. Similar reactions do not occur with Met under these conditions. Studies using murine macrophage-like J774A.1 cells demonstrate a greater peroxide-removing capacity in cells supplemented with SeMet, compared to nonsupplemented controls. Overall, these findings demonstrate that SeMet may play an important role in the catalytic removal of damaging peptide and protein oxidation products.     

Investigation of the functional role of tryptophan-22 in Escherichia coli dihydrofolate reductase by site-directed mutagenesis

We have applied site-directed mutagenesis methods to change the conserved tryptophan-22 in the substrate binding site of Escherichia coli dihydrofolate reductase to phenylalanine (W22F) and histidine (W22H). The crystal structure of the W22F mutant in a binary complex with the inhibitor methotrexate has been refined at 1.9-A resolution. The W22F difference Fourier map and least-squares refinement show that structural effects of the mutation are confined to the immediate vicinity of position 22 and include an unanticipated 0.4-A movement of the methionine-20 side chain. A conserved bound water-403, suspected to play a role in the protonation of substrate DHF, has not been displaced by the mutation despite the loss of a hydrogen bond with tryptophan-22. Steady-state kinetics, stopped-flow kinetics, and primary isotope effects indicate that both mutations increase the rate of product tetrahydrofolate release, the rate-limiting step in the case of the wild-type enzyme, while slowing the rate of hydride transfer to the point where it now becomes at least partially rate determining. Steady-state kinetics show that below pH 6.8, kcat is elevated by up to 5-fold in the W22F mutant as compared with the wild-type enzyme, although kcat/Km(dihydrofolate) is lower throughout the observed pH range. For the W22H mutant, both kcat and kcat/Km(dihydrofolate) are substantially lower than the corresponding wild-type values. While both mutations weaken dihydrofolate binding, cofactor NADPH binding is not significantly altered. Fitting of the kinetic pH profiles to a general protonation scheme suggests that the proton affinity of dihydrofolate may be enhanced upon binding to the enzyme. We suggest that the function of tryptophan-22 may be to properly position the side chain of methionine-20 with respect to N5 of the substrate dihydrofolate.     

Affinity labeling of dihydrofolate reductase with an antifolate glyoxal

Dihydrofolate reductases from different species contain several highly conserved arginines, some of which have been shown by x-ray crystallography to have their guanido groups near the p-aminobenzoyl glutamate moiety of enzyme-bound methotrexate. The orientation of one of these (Arg-52) appears to be completely reversed in comparing the crystal structures of Escherichia coli with Lactobacillus casei enzyme (Bolin, J. T., Filman, D. J., Matthews, D. A., Hamlin, R. C., and Kraut, J. (1982). J. Biol. Chem. 257, 13650-13662). We synthesized a novel antifolate containing a glyoxal group designed to react specifically with active-site guanido groups which are able to approach the p-aminobenzoyl carbonyl of methotrexate. The binding of this compound to the enzyme was competitive with dihydrofolate (DHF) in ordinary buffers. In borate buffer at pH 8.0 it inactivated dihydrofolate reductases from both E. coli and L. casei at similar maximum rates, while the chicken liver enzyme was more slowly inactivated. The inactivation was stoichiometric, paralleled the loss of the glyoxal chromophore, and showed saturation kinetics. Inhibitor binding and thus inactivation was enhanced by NADPH, while DHF protected the enzyme. This allowed calculation of the Kd for DHF which was found to be identical with its Km. The stoichiometrically inactivated enzyme displayed the 340-nm chromophore characteristic of 4-aminopteridines bound to dihydrofolate reductase confirming active-site labeling with normal orientation of the ligand. The ligand remained covalently bound to inactivated enzyme upon denaturation at low pH but dissociated at neutral pH. Computer graphic modeling of the crystal structures predicted reaction of Arg-31 but not Arg-52 in L. casei dihydrofolate reductase and of only Arg-52 in the E. coli enzyme. Purification of the CNBr fragments from the inactivated enzymes gave a single labeled peptide for each species. The particular peptide tagged in each case was unaffected by the presence of NADPH and was in excellent agreement with the crystallographic predictions.     

Identification of active site residues in Bradyrhizobium japonicum acetyl-CoA synthetase.

Acetyl-CoA synthetase (ACS) catalyses the activation of acetate to acetyl-CoA in the presence of ATP and CoA. The gene encoding Bradyrhyzobium japonicum ACS has been cloned, sequenced, and expressed in Escherichia coli. The enzyme comprises 648 amino acid residues with a calculated molecular mass of 71,996 Da. The recombinant enzyme was also purified from the transformed E. coli. The enzyme was essentially indistinguishable from the ACS of B. japonicum bacteroids as to the criteria of polyacrylamide gel electrophoresis and biochemical properties. Based on the results of database analysis, Gly-263, Gly-266, Lys-269, and Glu-414 were selected for site-directed mutagenesis in order to identify amino acid residues essential for substrate binding and/or catalysis. Four different mutant enzymes (G263I, G266I, K269G, and E414Q) were prepared and then subjected to steady-state kinetic studies. The kinetic data obtained for the mutants suggest that Gly-266 and Lys-269 participate in the formation of acetyl-AMP, whereas Glu-414 may play a role in acetate binding.     

Moritella cold-active dihydrofolate reductase: are there natural limits to optimization of catalytic efficiency at low temperature?

Adapting metabolic enzymes of microorganisms to low temperature environments may require a difficult compromise between velocity and affinity. We have investigated catalytic efficiency in a key metabolic enzyme (dihydrofolate reductase) of Moritella profunda sp. nov., a strictly psychrophilic bacterium with a maximal growth rate at 2 degrees C or less. The enzyme is monomeric (Mr=18,291), 55% identical to its Escherichia coli counterpart, and displays Tm and denaturation enthalpy changes much lower than E. coli and Thermotoga maritima homologues. Its stability curve indicates a maximum stability above the temperature range of the organism, and predicts cold denaturation below 0 degrees C. At mesophilic temperatures the apparent Km value for dihydrofolate is 50- to 80-fold higher than for E. coli, Lactobacillus casei, and T. maritima dihydrofolate reductases, whereas the apparent Km value for NADPH, though higher, remains in the same order of magnitude. At 5 degrees C these values are not significantly modified. The enzyme is also much less sensitive than its E. coli counterpart to the inhibitors methotrexate and trimethoprim. The catalytic efficiency (kcat/Km) with respect to dihydrofolate is thus much lower than in the other three bacteria. The higher affinity for NADPH could have been maintained by selection since NADPH assists the release of the product tetrahydrofolate. Dihydrofolate reductase adaptation to low temperature thus appears to have entailed a pronounced trade-off between affinity and catalytic velocity. The kinetic features of this psychrophilic protein suggest that enzyme adaptation to low temperature may be constrained by natural limits to optimization of catalytic efficiency.     

Resistance of Pediococcus cerevisiae to amethopterin as a consequence of changes in enzymatic activity and cell permeability. I. Dihydrofolate reductase, thymidylate synthetase and formyltetrahydrofolate synthetase in amethopterin-resistant and -sensitive strains of Pediococcus cerevisiae.

Pediococcus cerevisiae/AMr, resistant to amethopterin, possesses a higher dihydrofolate reductase (5, 6, 7, 8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) activity than the parent, a folate-permeable and thus amethopterin-susceptible strain and than the wild-type. The properties of dihydrofolate reductase from the three strains have been compared. Temperature, pH optima, heat stability, as well amethopterin binding did not reveal significant differences between the enzymes from the susceptible and resistant strains. The enzyme from the wild-type was 10 times more sensitive to inhibition by amethopterin and more susceptible to heat denaturation. The apparent Km values for dihydrofolate in enzymes from the three strains were in the range of 4.8--7.2 muM and for NADPH 6.5--8.0 muM. The amethopterin-resistant strain exhibited cross-resistance to trimethoprim and was about 40-fold more resistant to the latter than the sensitive parent and the wild-type. The resistance to trimethoprim appears to be a direct result of the increased dihydrofolate reductase activity. Inhibition of dihydrofolate reductase activity by this drug was similar in the three strains. 10--20 nmol caused 50% inhibition of 0.02 enzyme unit. Trimethoprim was about 10 000 times less effective inhibitor of dihydrofolate reductase than amethopterin. The cell extract of the AMr strain possessed a folate reductase activity three times higher than that of the sensitive strain. The activities of other folate-related enzymes like thymidylate synthetase and 10-formyltetrahydrofolate synthetase (formate: tetrahydrofolate ligase (ADP-forming), EC 6.3.4.3) were similar in the three strains studied.     

Thermodynamics and solvent effects on substrate and cofactor binding in Escherichia coli chromosomal dihydrofolate reductase

Chromosomal dihydrofolate reductase from Escherichia coli catalyzes the reduction of dihydrofolate to tetrahydrofolate using NADPH as a cofactor. The thermodynamics of ligand binding were examined using an isothermal titration calorimetry approach. Using buffers with different heats of ionization, zero to a small, fractional proton release was observed for dihydrofolate binding, while a proton was released upon NADP(+) binding. The role of water in binding was additionally monitored using a number of different osmolytes. Binding of NADP(+) is accompanied by the net release of ～5-24 water molecules, with a dependence on the identity of the osmolyte. In contrast, binding of dihydrofolate is weakened in the presence of osmolytes, consistent with "water uptake". Different effects are observed depending on the identity of the osmolyte. The net uptake of water upon dihydrofolate binding was previously observed in the nonhomologous R67-encoded dihydrofolate reductase (dfrB or type II enzyme) [Chopra, S., et al. (2008) J. Biol. Chem. 283, 4690-4698]. As R67 dihydrofolate reductase possesses a nonhomologous sequence and forms a tetrameric structure with a single active site pore, the observation of weaker DHF binding in the presence of osmolytes in both enzymes implicates cosolvent effects on free dihydrofolate. Consistent with this analysis, stopped flow experiments find betaine mostly affects DHF binding via changes in k(on), while betaine mostly affects NADPH binding via changes in k(off). Finally, nonadditive enthalpy terms when binary and ternary cofactor binding events are compared suggest the presence of long-lived conformational transitions that are not included in a simple thermodynamic cycle.     

Purification and properties of dihydrofolate reductase from methotrexate-sensitive and methotrexate-resistant Chinese hamster ovary cells.

We have previously described methotrexate-resistant Chinese hamster ovary cells which appear to contain normal levls of a structurally altered dihydrofolate reductase (EC 1.5.1.3) (Flintoff, W.F., Davidson, S.V., and Siminovitch, L. (1976) Somatic Cell Genet.2,245-261). By selecting for increased resistance form these class I cells, class III resistant cells were isolated which appeared to possess an increased activity of the altered enzyme. In the report, we describe the purification and several properties of the reductase from wild-type cells, two independently selected class I cells, and class III resistant cell. The reductases from wild-type and resistant cells had similar specific activities using folate and dihydrofolate as substrates, and similar molecular weights as determined by sodium dodecyl sulfate gel electrophoresis. The mutant enzymes, however, were about six- to eight-fold more resistant to inhibition by methotrexate than the wild-type enzyme, suggesting a decreased affinity of the mutant reductases to methotrexate-binding. Small differences between various enzymes were also seen in other physicochemical properties such as pH optima and Km values for folate, and in their heat stabilities, which suggest that different structural alterations may lead to the same mutant phenotype. As expected from earlier studies with crude extracts, class III cells did produce a higher (about 10-fold) yield of the reductase than the class I or wild-type cells.     

The purification and properties of the trimethoprim-resistant dihydrofolate reductase mediated by the R-factor, R388.

The R-factor R388 mediates the production of a trimethoprim-resistant dihydrofolate reductase. This enzyme has a different molecular weight and pH profile to the trimethoprim-sensitive enzyme of the Escherichia coli host. The R-factor mediated enzyme was separated completely from the host E. coli enzyme by DEAE-cellulose ion-exchange chromatography. The purified R-factor enzyme was about 20 000 times less susceptible to trimethoprim than the E. coli enzyme and although it was inhibited competitively by trimethoprim, its inhibitor constant (Ki) was 20 000 times greater than that of the host enzyme. The R388 and E. coli enzymes also differed in their substrate specificity requirements. In addition, the R388 enzyme suprisingly conferred high level resistance to the broad spectrum dihydrofolate reductase inhibitor, amethopterin. The possible origins of the R388 enzyme are discussed.