Malaria is caused by the protozoan parasite, Plasmodium and inflicts much suffering in the developing world with more than a million deaths recorded each year. In addition, a significant number of cases of malaria are imported into nonmalarious areas by tourists, military personnel, and migrant workers. The prospects for control of malaria by chemotherapy have been seriously impeded by the emergence of drug-resistant parasite strains. This accentuates the need to study the biochemistry of this parasite so that potential target for chemotherapy can be identified. Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in the de novo synthesis of deoxyribonucleotides by the direct reduction of the corresponding ribonucleotides. The important role of RNR in DNA synthesis and cell division makes this enzyme an excellent target for chemotherapy. Several different classes of inhibitors targeted against RNR for cancer and viral chemotherapy already exist. Critical to the development of a chemotherapeutic agent directed against RNR from the malaria parasite is a detailed understanding of the biochemistry of this complex enzyme. The proposed investigation is an attempt to characterize ribonucleotide reductase from plasmodium falciparum (PF). Specifically we aim to: 1) Purify the native enzyme from PF culture lysates by chromatographic methods and assess its catalytic activity. 2) Test different classes of known mammalian and viral RNR inhibitors for antimalarial activity by measuring the effects on [3H]-hypoxanthine incorporation into nucleic acids. The kinetics of inhibition (Ki) of these inhibitors will be studied with native and/or recombinant enzyme preparation. 3) Overexpress and purify the recombinant RNR subunits in E. coli and/or baculovirus expression systems. Compare the activities of the recombinant and native RNR. 4) Study kinetic properties and the allosteric regulation of the PF RNR. 5) Study the in vivo expression of RNR subunit genes in asexual blood stages upon addition of DNA damaging or modifying agents by Northern blot analysis and nuclear run-on assay.
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