Expression and Characterization of Recombinant Human-Derived Pneumocystis carinii Dihydrofolate Reductase
AUTOR(ES)
Ma, Liang
FONTE
American Society for Microbiology
RESUMO
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 Km values for dihydrofolate and NADPH were 2.7 ± 0.3 and 14.0 ± 4.3 μM, 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 Ki values of 0.28 ± 0.08 and 0.065 ± 0.005 μM, respectively, while trimetrexate and methotrexate were potent inhibitors, with Ki 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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=101608Documentos Relacionados
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