ABSTRACT
Pertussis toxin is produced by the causative agent of whooping cough, Bordetella pertussis, and is an adenosine diphosphate (ADP)-ribosyltransferase capable of covalently modifying and thereby inactivating many eukaryotic G proteins involved in cellular metabolism. The toxin is a principal determinant of virulence in whooping cough and is a primary candidate for an acellular pertussis vaccine, yet it is unclear whether the ADP-ribosyltransferase activity is required for both pathogenic and immunoprotective activities. A B. pertussis strain that produced an assembled pertussis holotoxin with only 1 percent of the ADP-ribosyltransferase activity of the native toxin was constructed and was found to be deficient in pathogenic activities associated with B. pertussis including induction of leukocytosis, potentiation of anaphylaxis, and stimulation of histamine sensitivity. Moreover, this mutant strain failed to function as an adjuvant and was less effective in protecting mice from intracerebral challenge infection. These data suggest that the ADP-ribosyltransferase activity is necessary for both pathogenicity and optimum immunoprotection. These findings bear directly on the design of a nontoxic pertussis vaccine.
Subject(s)
Bordetella pertussis/immunology , Pentosyltransferases/metabolism , Pertussis Toxin , Virulence Factors, Bordetella/metabolism , ADP Ribose Transferases , Adjuvants, Immunologic , Anaphylaxis/etiology , Animals , Antigens/immunology , Bordetella pertussis/enzymology , Bordetella pertussis/genetics , Codon , Drug Tolerance , Histamine/pharmacology , Immunization , Leukocytosis/etiology , Macromolecular Substances , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mutation , Ovalbumin/immunology , Virulence Factors, Bordetella/genetics , Virulence Factors, Bordetella/immunologyABSTRACT
Crystalline R67 dihydrofolate reductase (DHFR) is a dimeric molecule with two identical 78 amino acid subunits, each folded into a beta-barrel conformation. The outer surfaces of the three longest beta strands in each protomer together form a third beta barrel having six strands at the subunit interface. A unique feature of the enzyme structure is that while the intersubunit beta barrel is quite regular over most of its surface, an 8-A "gap" runs the full length of the barrel, disrupting potential hydrogen bonds between beta-strand D in subunit I and the adjacent corresponding strand of subunit II. It is proposed that this deep groove is the NADPH binding site and that the association between protein and cofactor is modulated by hydrogen-bonding interactions along one face of this antiparallel beta-barrel structure. A hypothetical model is proposed for the R67 DHFR-NADPH-folate ternary complex that is consistent with both the known reaction stereoselectivity and the weak binding of 2,4-diamino inhibitors to the plasmid-specified reductase. Geometrical comparison of this model with an experimentally determined structure for chicken DHFR suggests that chromosomal and type II R-plasmid specified enzymes may have independently evolved similar catalytic machinery for substrate reduction.
Subject(s)
Escherichia coli/genetics , R Factors , Tetrahydrofolate Dehydrogenase/genetics , Trimethoprim/pharmacology , Drug Resistance, Microbial , Escherichia coli/drug effects , Escherichia coli/enzymology , Models, Molecular , Protein Conformation , Tetrahydrofolate Dehydrogenase/metabolism , X-Ray DiffractionABSTRACT
Dihydrofolate reductase specified by plasmid R483 from a trimethoprim-resistant strain of Escherichia coli has been purified 2,000-fold to homogeneity using dye-ligand chromatography, gel filtration, and polyacrylamide gel electrophoresis. The protein migrated as a single band on nondenaturing polyacrylamide gel electrophoresis and had a specific activity of 250 mumol/mg min(-1). The molecular weight was estimated to be 32,000 by gel filtration and 39,000 by Ferguson analysis of polyacrylamide gel electrophoresis. When subjected to electrophoresis in the presence of sodium dodecyl sulfate, the protein migrated as a single 19,000-molecular weight species, a fact that suggests that the native enzyme is a dimer of similar or identical subunits. Antibody specific for R483-encoded dihydrofolate reductase did not cross-react with dihydrofolate reductase encoded by plasmid R67, T4 phage, E. coli RT500, or mouse L1210 leukemia cells. The amino acid sequence of the first 34 NH2-terminal residues suggests that the R483 plasmid dihydrofolate reductase is more closely related to the chromosomal dihydrofolate reductase than is the enzyme coded by plasmid R67.
Subject(s)
Plasmids , Tetrahydrofolate Dehydrogenase/analysis , Amino Acid Sequence , Escherichia coli/enzymology , Kinetics , Macromolecular SubstancesABSTRACT
The alpha epimers of pyridine nucleotides are almost totally inactive as reductants in dehydrogenase reactions. In contrast, the R plasmid R67-specified dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) isolated from trimethoprim-resistant Escherichia coli utilized alpha-NADPH and alpha-NADH in addition to the "normal" beta-epimers. The enzymes from bacterial and mammalian sources used only beta-NADPH and beta-NADH. THe Km value for alpha-NADPH (16 microM) was 4-fold greater than that for beta-NADPH (4 microM), while the maximal velocity of the alpha-NADPH-catalyzed reaction was 70% of that seen with the beta-NADPH. beta-NADP+ and alpha-NADP+ were competitive inhibitors of the R67 enzyme. Pyridine nucleotide analogues such as deamino- and acetyl-NADPH were used readily by bacterial, plasmid, and mammalian enzymes, whereas thio-NADPH was used only by the plasmid enzyme. These data suggest that the enzyme from R plasmid R67 possesses a pyridine nucleotide binding site different from that of other dihydrofolate reductases and dehydrogenases.
Subject(s)
Bacteria/enzymology , Liver/enzymology , NADP/analogs & derivatives , NADP/metabolism , NAD/metabolism , Plasmids , Tetrahydrofolate Dehydrogenase/genetics , Animals , Clostridium/enzymology , Escherichia coli/enzymology , Isomerism , Kinetics , Lacticaseibacillus casei/enzymology , Rats , Species Specificity , Substrate Specificity , Tetrahydrofolate Dehydrogenase/metabolismABSTRACT
Resistance to inhibitors of dihydrofolate reductase arises from a variety of mechanisms involving enzyme alteration, cellular impermeability, enzyme overproduction, inhibitor modification, and loss of binding capacity. The mechanism of greatest clinical importance is the production of plasmid-encoded, trimethoprim-resistant forms of dihydrofolate reductase. At least two different types of these enzymes have been documented. The trimethoprim-resistant reductases differ from all other dihydrofolate reductases in molecular weight, subunit structure, kinetic properties, and binding of inhibitors. Colony hybridization techniques, developed for the detection of plasmid DNA coding for trimethoprim-resistant reductases, enable researchers to evaluate the prevalence and distribution of plasmid-borne resistance. Preliminary results obtained with a series of enzymatically characterized clinical isolates suggest that the colony hybridization technique may provide a convenient epidemiological tool for monitoring the dissemination of plasmid-borne resistance to trimethoprim.
Subject(s)
Bacteria/drug effects , Trimethoprim/pharmacology , Autoradiography , Bacteria/genetics , Bacterial Infections/microbiology , DNA, Bacterial/metabolism , Drug Resistance, Microbial , Escherichia coli/drug effects , Folic Acid Antagonists , Genes, Bacterial/drug effects , Humans , R FactorsSubject(s)
Anti-Bacterial Agents/history , Folic Acid Antagonists/history , Pyrimidines/history , Aminopterin/pharmacology , Animals , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Escherichia coli/enzymology , Folic Acid/metabolism , Folic Acid Antagonists/pharmacology , Folic Acid Antagonists/therapeutic use , History, 20th Century , Pyrimidines/pharmacology , Pyrimidines/therapeutic use , Thymidine/biosynthesisABSTRACT
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.
Subject(s)
Liver/enzymology , Tetrahydrofolate Dehydrogenase , Amino Acid Sequence , Amino Acids/analysis , Animals , Cattle , Chickens , Kinetics , Leukemia L1210/enzymology , Mice , Molecular Weight , Species Specificity , Substrate Specificity , Swine , Tetrahydrofolate Dehydrogenase/isolation & purification , Tetrahydrofolate Dehydrogenase/metabolismABSTRACT
Dihydrofolate reductase, specified by the type II plasmid of a trimethoprim-resistant Escherichia coli, was purified 40-fold to homogeneity using a combination of gel filtration, DEAE-Sephacel chromatography, and hydrophobic chromatography. The final product shows a single protein band on polyacrylamide gel electrophoresis and has a specific activity of 1.0 unit/mg. The molecular weight of the purified enzyme is 36,000 as determined both by gel filtration and Ferguson analysis of polyacrylamide gel electrophoresis. In contrast, a single polypeptide with a molecular weight of 8,500 was observed on sodium dodecyl sulfate-gel electrophoresis. These experiments suggest that, unlike any bacteria or vertebrate dihydrofolate reductase previously examined, the type II R plasmid reductase is a tetramer composed of four identical subunits. A partial amino acid sequence determination shows no heterogeneity of the subunits and also no clear homology with any reductase sequence previously reported.
Subject(s)
Escherichia coli/enzymology , Plasmids , Tetrahydrofolate Dehydrogenase/isolation & purification , Amino Acid Sequence , Escherichia coli/drug effects , Macromolecular Substances , Molecular Weight , Tetrahydrofolate Dehydrogenase/metabolism , Transformation, Genetic , Trimethoprim/pharmacologyABSTRACT
Members of the genus Neisseria are relatively nonsusceptible to trimethoprim, an inhibitor of dihydrofolate reductase. For example, the minimal inhibitory concentration (MIC) of trimethoprim for N. gonorrhoeae ranges from 2 to 70 mug/ml, whereas the MIC for Escherichia coli is 0.2 mug/ml or less. In an effort to understand this difference, dihydrofolate reductase was partially purified from five Neisseria species and compared with the enzyme from E. coli. N. gonorrhoeae dihydrofolate reductase was similar to that from E. coli in molecular weight (18,000) and affinity for the substrates reduced nicotinamide adenine dinucleotide phosphate and dihydrofolate (K(m) = 13 and 8 muM, respectively). However, the gonococcal enzyme had a decreased affinity for trimethoprim, with an apparent K(i) of 45 x 10(-9) M, some 30-fold greater than the E. coli value of 1.2 x 10(-9) M. These enzymes also differed in their isoelectric points and pH activity profiles. Within the genus Neisseria, the dihydrofolate reductase isolated from N. meningitidis and N. lactamica resembled the N. gonorrhoeae enzyme, and only small differences were detected for the N. flavescens and Branhamella catarrhalis dihydrofolate reductases. These data indicate that the relatively poor affinity of trimethoprim for the dihydrofolate reductase from these organisms may be largely responsible for the relative nonsusceptibility of Neisseria sp. to trimethoprim. The contribution of other resistance mechanisms to the overall nonsusceptibility was assessed. Strains of N. gonorrhoeae with altered cell envelope permeability had MIC values less than twofold different from those of isogenic wild-type strains. Also, a direct relationship was observed between the affinity of trimethoprim analogs for gonococcal dihydrofolate reductase and the MIC of these compounds for the gonococcus. These observations suggest that the cell envelope of N. gonorrhoeae is not impermeable to trimethoprim. Changes in the amount of dihydrofolate reductase activity could cause alterations in the susceptibility of the gonococcus to trimethoprim, as demonstrated with N. gonorrhoeae strains selected for trimethoprim resistance after chemical mutagenesis. However, the level of dihydrofolate reductase activity in wild-type N. gonorrhoeae was similar to that of E. coli, indicating that the difference in the susceptibility of these organisms is not due to greater amounts of enzyme in N. gonorrhoeae.
Subject(s)
Neisseria/enzymology , Tetrahydrofolate Dehydrogenase/metabolism , Bacteria/drug effects , Folic Acid Antagonists , Mutation/drug effects , Neisseria gonorrhoeae/genetics , Trimethoprim/pharmacologyABSTRACT
A combination of affinity column chromatography and preparative gel electrophoresis has been used to purify to homogeneity the two isozymes of dihydrofolate reductase from a trimethoprim-resistant strain of Escherichia coli B (RT 500). These enzyme forms are noninterconvertible and are present in crude cell lysates, but other electrophoretic species can be generated durng purification if sulfhydryl-protecting agents, such as dithiothreitol, are not present. The two isozymes, numbered form 1 and form 2 with respect to their decreasing electrophoretic mobilities, have similar molecular weights (18 500), molecular radii (21 A), and apparent Km values for reduced nico inamide adenin- dinucleotide (NADH) and NADH phosphate (NADPH). Both forms contain 2 mol of sulfhydryl/mol of enzyme which can be oxidized to intramolecular disulfide bonds. However, forms 1 and 2 differ physically in their electrophoretic mobility and isoelectric point and kinetically in their pH-activity profile, specific activity, Km for dihydrofolate, and their affinity toward a number of inhibitors.
Subject(s)
Escherichia coli/enzymology , Isoenzymes/isolation & purification , Tetrahydrofolate Dehydrogenase/isolation & purification , Hydrogen-Ion Concentration , Isoenzymes/metabolism , Kinetics , Molecular Weight , Tetrahydrofolate Dehydrogenase/metabolismABSTRACT
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 (Sköld, 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.
Subject(s)
Escherichia coli/enzymology , Extrachromosomal Inheritance/drug effects , Isoenzymes/biosynthesis , Plasmids/drug effects , Protein Biosynthesis/drug effects , Tetrahydrofolate Dehydrogenase/biosynthesis , Transcription, Genetic/drug effects , Trimethoprim/pharmacology , Chromosomes, Bacterial/enzymology , Drug Resistance, Microbial , Escherichia coli/drug effects , Hydrogen-Ion Concentration , Kinetics , Methotrexate/pharmacology , NADP , Phenotype , Species SpecificityABSTRACT
The determination of the amino acid sequence of the dihydrofolate reductase from Escherichia coli RT500 is described. The sequence, comprising 159 residues, has been derived from automatic sequencing of the intact protein in conjunction with manual sequencing of lysine-blocked tryptic peptides, Staphylococcus aureus protease peptides, and alpha-lytic protease peptides. Comparison of the sequence with that of the dihydrofolate reductase from a methotrexate-resistant strain of E. coli (MB1428) shows that 145 of the residues are identical. The distribution of the differences along the length of the molecule is discussed.
Subject(s)
Escherichia coli/enzymology , Tetrahydrofolate Dehydrogenase , Trimethoprim/pharmacology , Amino Acid Sequence , Amino Acids/analysis , Cyanogen Bromide , Drug Resistance, Microbial , Escherichia coli/drug effects , Peptide Fragments/analysis , Peptide Hydrolases , Staphylococcus aureus/enzymology , Tetrahydrofolate Dehydrogenase/metabolism , TrypsinABSTRACT
Dihydrofolate reductase from Escherichia coli exists as two species, which show large differences in their affinities for trimethoprim and for pyrimethamine. The two species are present in approximately equal proportions. Each possesses one binding site per mol with dissociation constants (KD) of 14 and 1400 nM, respectively, for the binding of trimethoprim in the binary complex, and of 5 and 47 nM for the pyrimethamine binary complex. In the formation of the ternary complex with NADPH, trimethoprim bound to dihydrofolate reductase as if all the enzyme existed as a single species with a KD for trimethoprim of 1.9 nM. Formation of the trimethoprim NADPH ternary complex thus involves strong cooperative effects, and interconversion of the two species. The binding of pyrimethamine in the ternary complex was indistinguishable from its binding in the binary complex, showing neither the cooperative effects, nor the interconversion of the two species observed with trimethoprim. The species with a low KD for trimethoprim and pyrimethamine could be isolated by selective proteolysis. It was quite stable, but could be converted to a mixture of the original species via formation of the ternary complex with trimethoprim, as predicted from the binding data. The results are interpreted in terms of a model in which the two species can be interconverted only via the formation of a common ternary complex, which can be formed after the binding of trimethoprim or dihydrofolate, but not pyrimethamine. The model is shown to be consistent with all data from the measurements of both binding and inhibition by both ligands.
Subject(s)
Escherichia coli/enzymology , Isoenzymes/metabolism , Tetrahydrofolate Dehydrogenase/metabolism , Binding Sites , Kinetics , Mathematics , Pronase , Protein Binding , Pyrimethamine/pharmacology , Trimethoprim/pharmacologyABSTRACT
Dihydrofolate reductase has been purified 40-fold to apparent homogeneity from a trimethoprim-resistant strain of Escherichia coli (RT 500) using a procedure that includes methotrexate affinity column chromatography. Determinations of the molecular weight of the enzyme based on its amino acid composition, sedimentation velocity, and sodium dodecyl sulfate gel electrophoresis gave values of 17680, 17470 and 18300, respectively. An aggregated form of the enzyme with a low specific activity can be separated from the monomer by gel filtration; treatment of the aggregate with mercaptoethanol or dithiothreitol results in an increase in enzymic activity and a regeneration of the monomer. Also, multiple molecular forms of the monomer have been detected by polyacrylamide gel electrophoresis. The unresolved enzyme exhibits two pH optima (pH 4.5 and pH 7.0) with dihydrofolate as a substrate. Highest activities are observed in buffers containing large organic cations. In 100 mM imidazolium chloride (pH 7), the specific activity is 47 mumol of dihydrofolate reduced per min per mg at 30 degrees. Folic acid also serves as a substrate with a single pH optimum of pH 4.5. At this pH the Km for folate is 16 muM, and the Vmax is 1/1000 of the rate observed with dihydrofolate as the substrate. Monovalent cations (Na+, K+, Rb+, and Cs+) inhibit dihydrofolate reductase; at a given ionic strength the degree of inhibition is a function of the ionic radius of the cation. Divalent cations are more potent inhibitors; the I50 of BaCl2 is 250 muM, as compared to 125 mM for KCl. Anions neither inhibit nor activate the enzyme.
Subject(s)
Escherichia coli/enzymology , Tetrahydrofolate Dehydrogenase/isolation & purification , Amino Acids/analysis , Chromatography, Affinity , Hydrogen-Ion Concentration , Isoenzymes/isolation & purification , Isoenzymes/metabolism , Kinetics , Macromolecular Substances , Methotrexate , Molecular Weight , Osmolar Concentration , Potassium Chloride/pharmacology , Protein Conformation , Sodium Chloride/pharmacology , Tetrahydrofolate Dehydrogenase/metabolismABSTRACT
Rich media support the growth of bacteria in the presence of concentrations of sulfonamides and diaminopyrimidines that are highly inhibitory when the organisms are grown on minimal media. Many such rich media can be made more suitable for susceptibility testing by the incorporation of lysed horse blood. Harper and Cawston characterized the active substance, Harper-Cawston factor (HCF), and later studies indicated it to be a protein. It has now been identified as thymidine phosphorylase. The identification follows from the identical purification pattern of HCF and thymidine phosphorylase activities from horse blood to a high degree of purity. Blood of goats, sheep, oxen, geese, chickens, cows, dogs, rats, and humans had neither biological activity. The identification of HCF as thymidine phosphorylase is consistent with the earlier findings of Koch and Burchall (1971) that most of the interfering effects of rich media could be accounted for by their thymidine contents, and that thymidine is much more active in this respect than is thymine.
Subject(s)
Bacteria/enzymology , Drug Resistance, Microbial , Extrachromosomal Inheritance , Pentosyltransferases/physiology , Pyrimidines/pharmacology , Sulfonamides/pharmacology , Thymidine Phosphorylase/physiology , Animals , Culture Media , Horses/metabolism , Microbial Sensitivity Tests/methods , Thymidine Phosphorylase/isolation & purificationSubject(s)
Cyclizine/metabolism , Histamine H1 Antagonists/metabolism , Piperazines/metabolism , Polymyxins/metabolism , Protein Binding , Amino Acids/pharmacology , Aminobutyrates/pharmacology , Animals , Choline/pharmacology , Coloring Agents , Dealkylation , Dogs , Female , Glutathione/pharmacology , Humans , Kidney/metabolism , Liver/metabolism , Lung/metabolism , Male , Mice , Myocardium/metabolism , Rats , Species Specificity , Spleen/metabolism , Time Factors , TritiumSubject(s)
Sulfamethoxazole/metabolism , Trimethoprim/metabolism , Amino Acid Sequence , Animals , Anura , Bacteria/enzymology , Binding Sites , Drug Combinations , Fishes , Folic Acid/metabolism , Folic Acid Antagonists , Guinea Pigs , Humans , Hydrogen-Ion Concentration , Kinetics , Oxidoreductases/classification , Oxidoreductases/metabolism , Protein Binding , Rabbits , Rats , Species Specificity , Sulfamethoxazole/pharmacology , Tetrahydrofolate Dehydrogenase/metabolism , Tetrahydrofolates/metabolism , Trimethoprim/pharmacology , TurtlesSubject(s)
Polymyxins/metabolism , Aminobutyrates/pharmacology , Animals , Brain Chemistry , Choline/pharmacology , Chromatography, Thin Layer , Cysteine/pharmacology , Escherichia coli/drug effects , Glutathione/pharmacology , Kidney/analysis , Liver/analysis , Lung/analysis , Male , Methionine/pharmacology , Mice , Mice, Inbred Strains , Myocardium/analysis , Polymyxins/analysis , Polymyxins/blood , Polymyxins/pharmacology , Protein Binding , Threonine/pharmacology , Time Factors , TritiumSubject(s)
Tetrahydrofolate Dehydrogenase , Animals , Anura , Biochemical Phenomena , Biochemistry , Escherichia coli/enzymology , Fishes , Folic Acid Antagonists , Humans , Hydrogen-Ion Concentration , Kinetics , Liver/enzymology , Malaria/enzymology , Methotrexate/pharmacology , Nephropidae/enzymology , Proteus vulgaris/enzymology , Pyridines/pharmacology , Pyrimidines/pharmacology , Quinazolines/pharmacology , Rana pipiens , Rats , Staphylococcus/enzymology , Taenia/enzymology , Triazines/pharmacology , Trimethoprim/pharmacology , TurtlesABSTRACT
The ability of a potent dihydrofolate reductase inhibitor, trimethoprim, to inhibit the growth of Escherichia coli B in vitro is dependent on the composition of the medium in which the cells are grown. The inhibition observed in minimal broth could be partially reversed by the addition of thymidine, ribonucleosides, amino acids, and vitamins. No reversal occurred in the absence of thymidine. In a number of commercially prepared media, the inhibitory activity of trimethoprim correlated inversely with the amount of thymidine found to be present by microbiological assay. The significance of these findings for the routine testing of new, synthetic antibacterial agents is discussed.