Abstract
Trypanosoma brucei is the causative agent for African sleeping sickness. We have made in vitro and in vivo studies on the allosteric regulation of the trypanosome ribonucleotide reductase, a key enzyme in the production of dNTPs needed for DNA synthesis. Results with the isolated recombinant trypanosome ribonucleotide reductase showed that dATP specifically directs pyrimidine ribonucleotide reduction instead of being a general negative effector as in other related ribonucleotide reductases, whereas dTTP and dGTP directed GDP and ADP reduction, respectively. Pool measurements of NDPs, NTPs, and dNTPs in the cultivated bloodstream form of trypanosomes exposed to deoxyribonucleosides or inhibited by hydroxyurea confirmed our in vitro allosteric regulation model of ribonucleotide reductase. Interestingly, the trypanosomes had extremely low CDP and CTP pools, whereas the dCTP pool was comparable with that of other dNTPs. The trypanosome ribonucleotide reductase seems adapted to this situation by having a high affinity for the CDP/UDP-specific effector dATP and a high catalytic efficiency, Kcat/Km, for CDP reduction. Thymidine and deoxyadenosine were readily taken up and phosphorylated to dTTP and dATP, respectively, the latter in a nonsaturating manner. This uncontrolled uptake of deoxyadenosine strongly inhibited trypanosome proliferation, a valuable observation in the search for new trypanocidal nucleoside analogues.
Highlights
Trypanosoma brucei is an African unicellular eukaryote that lives extracellularly in the mammalian bloodstream and central nervous system as well as in the guts and salivary glands of tsetse flies
With dATP [5, 6] or ATP bound to the specificity sites, CDP and UDP are reduced, whereas dTTP and dGTP gives GDP and ADP reduction, respectively
We found that the deoxyribonucleotide metabolism of T. brucei in many aspects is unique, in the allosteric regulation of ribonucleotide reductase and in the properties of the deoxyribonucleoside kinases
Summary
Determination of NTP and dNTP Pools by HPLC1—The bloodstream form of T. brucei (strain 221) (kindly supplied by Fred Opperdoes, ICP, Trop 7439, avenue Hippocrate 74, B-1200, Brussels, Belgium) was maintained at 37 °C and 7.2% CO2 in Hirumi’s modified Iscoves Medium (HMI)-9 medium [14] lacking thymidine and Serum Plus but containing 10% fetal calf serum. Protein R1 (10 g) was incubated for 5 min in 50 mM Tris-HCl, pH 7.6, 50 mM KCl, 10 mM DTT, 6.4 M MgCl2, 4.5 M tritiated dGTP, and varying amounts of competing cold nucleotides In those cases where the total nucleotide level exceeded 3 mM, the MgCl2 concentration was increased to constitute twice the level of nucleotides. The substrates used were [5-3H]CDP (Amersham Pharmacia Biotech), [2-14C]UDP (kindly supplied by Peter Reichard, Karolinska Institute, S-171 77 Stockholm, Sweden), [8-14C]GDP (Moravek Biochemicals), and [8-14C]ADP (NEN Life Science Products) They were diluted with unlabeled nucleotides from Sigma into specific activities of 37,000, 3,320, 3,570, and 1,400 cpm/nmol, respectively. A 1 M solution of NH4HCO3, pH 8.9, was added to the supernatant to a final concentration of 50 mM, and the products were separated from the substrates by boronate affinity chromatography as described for the dNTP pool determination. Coformycin (Calbiochem) was stored as a 3 mM aqueous solution at Ϫ20 °C
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