Cloning, Expression, and Characterization of Babesia gibsoni Dihydrofolate Reductase-Thymidylate Synthase: Inhibitory Effect of Antifolates on Its Catalytic Activity and Parasite Proliferation
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Date
2008Author
Aboge, Gabriel O
Terkawi, Mohamad A
Goo, Youn-Kyoung
Nishikawa, Yoshifumi
Sunaga, Fujiko
Namikawa, Kuzuhiko
Tsuji, Naotoshi
Igarashi, Ikuo
Suzuki, Hiroshi
Fujisaki, Kozo
Xuan, Xuenan
Type
ArticleLanguage
enMetadata
Show full item recordAbstract
Dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a well-validated antifolate drug target in certain pathogenic apicomplexans, but not in the genus Babesia, including Babesia gibsoni. Therefore, we isolated, cloned, and expressed the wild-type B. gibsoni dhfr-ts gene in Escherichia coli and evaluated the inhibitory effect of antifolatesonits enzymeactivity,aswellasoninvitroparasitegrowth.Thefull-lengthgene
consists of a 1,548-bp open reading frame encoding a 58.8-kDa translated peptide containing DHFR and TS domains linked together in a single polypeptide chain. Each domain contained active-site amino acid residues responsible for the enzymatic activity. The expressed soluble recombinant DHFR-TS protein was approximately
57 kDa after glutathione S-transferase (GST) cleavage, similar to an approximately 58-kDa native enzyme identified from the parasite merozoite. The non-GST fusion recombinant DHFR enzyme revealed K m values of 4.70 0.059 (mean standard error of the mean) and 9.75 1.64M for dihydrofolic acid
(DHF) and NADPH, respectively. Methotrexate was a more-potent inhibitor of the enzymatic activity (50% inhibition concentration [IC50] 68.6 5.20 nM) than pyrimethamine (IC50 55.0 2.08M) and trimethoprim (IC50 50 12.5M). Moreover, the antifolates’ inhibitory effects on DHFR enzyme activity paralleled their inhibition of the parasite growth in vitro, indicating that the B. gibsoni DHFR
could be a model for studying antifolate compounds as potential drug candidates. Therefore, theB.gibsoni DHFR-TS is a molecular antifolate drug target.
Citation
Antimicrobial agents and chemotherapy, Nov. 2008, Vol. 52, No. 11, p. 4072–4080Publisher
Department of Public Health Pharmacology and Toxicology