title: Purification and properties of recombinant Pneumocystis carinii dihydrofolate reductase.
abstract: Pneumocystis carinii dihydrofolate reductase (DHFR) expressed in Escherichia coli was purified to homogeneity in a single step using methotrexate-Sepharose affinity chromatography. The purified enzyme migrated as a single 24-kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequence of the first 26 amino acids from the N-terminus of the purified enzyme was in accord with that predicted from the DNA sequence. The enzyme showed a broad pH optimum with maximum activity over the pH range 6 to 7. The enzyme was activated by salts, with maximal twofold activation at 50 to 150 mM KCl and 50 to 200 mM NaCl. Urea at 2.5 M also increased the enzyme activity twofold. Kinetic analysis of the purified enzyme revealed that the Km values for dihydrofolate and NADPH were 1.8 and 1.4 microM, respectively, and that the kcat was 70 s-1. Inhibition studies verified that trimethoprim and pyrimethamine were poor inhibitors of P. carinii DHFR and showed little selectivity over the human DHFR. Trimetrexate and piritrexim were much more potent inhibitors of the P. carinii enzyme, but these inhibitors are also potent inhibitors of human DHFR.
authors.affiliation: Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand.
authors.forename: W
authors.initials: W
authors.lastname: Sirawaraporn
authors.forename: J C
authors.initials: JC
authors.lastname: Edman
authors.forename: D V
authors.initials: DV
authors.lastname: Santi
mesh_terms: D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D011010:Pneumocystis / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D013762:Tetrahydrofolate Dehydrogenase