title: Expression and characterization of recombinant human-derived Pneumocystis carinii dihydrofolate reductase.
abstract: Dihydrofolate reductase (DHFR) is the target of trimethoprim (TMP), which has been widely used in combination with sulfa drugs for treatment and prophylaxis of Pneumocystis carinii pneumonia. While the rat-derived P. carinii DHFR has been well characterized, kinetic studies of human-derived P. carinii DHFR, which differs from rat-derived P. carinii DHFR by 38% in amino acid sequence, have not been reported to date. Here we report on the expression and kinetic characterization of the recombinant human-derived P. carinii DHFR. The 618-bp coding sequence of the human-derived P. carinii DHFR gene was expressed in Escherichia coli. As determined by sodium dodecyl sulfate-polyacrylamide gel eletrophoresis, the purified enzyme had a molecular mass of 25 kDa, consistent with that predicted from the DNA sequence. Kinetic analysis showed that the K(m) values for dihydrofolate and NADPH were 2.7 +/- 0.3 and 14.0 +/- 4.3 microM, respectively, which are similar to those reported for rat-derived P. carinii DHFR. Inhibition studies revealed that both TMP and pyrimethamine were poor inhibitors of human-derived P. carinii DHFR, with K(i) values of 0.28 +/- 0.08 and 0.065 +/- 0.005 microM, respectively, while trimetrexate and methotrexate were potent inhibitors, with K(i) values of 0.23 +/- 0.03 and 0.016 +/- 0.004 nM, respectively. The availability of purified recombinant enzyme in large quantities should facilitate the identification of antifolate inhibitors with greater potency and higher selectivity for human-derived P. carinii DHFR.
authors.affiliation: Critical Care Medicine Department, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: L
authors.initials: L
authors.lastname: Ma
authors.forename: J A
authors.initials: JA
authors.lastname: Kovacs
mesh_terms: D001667:Binding, Competitive; D002384:Catalysis; D004926:Escherichia coli; D005493:Folic Acid Antagonists / Q000494:pharmacology; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D011010:Pneumocystis / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014162:Transfection
chemical_list: D005493:Folic Acid Antagonists; D011994:Recombinant Proteins; D013762:Tetrahydrofolate Dehydrogenase