Allosteric Regulation of Trypanosoma bruceiRibonucleotide Reductase Studied in Vitro and in Vivo

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, K cat /K m , 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.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. Residing in its mammalian host, it causes a fatal disease called sleeping sickness. There is an urgent need to find new chemotherapy against this disease because the current ones are limited by toxicity as well as an increasing resistance among the trypanosomes (1).Ribonucleotide reductase (2) is a key enzyme in DNA synthesis because it catalyzes the reduction of ribonucleotides to deoxyribonucleotides, a reaction assisted by a protein-bound or a 5Ј-deoxyadenosyl cobalamin-derived free radical. Primarily based on the nature of this radical, the ribonucleotide reductases are divided into three classes where most of the eukaryotic and some of the prokaryotic ones belong to class I. The enzymes in this class are heterodimers formed from two large (R1) and two small (R2) polypeptides. The R1 protein binds substrates and allosteric effectors, whereas the R2 protein contains a tyrosyl radical essential for enzyme activity. The radical can be scavenged by the one-electron reductant hydroxyurea, a drug that has been in clinical use against leukemias (3).Except those from the Herpesviridae family (4), all ribonucleotide reductases studied are allosterically regulated. The nonviral class I enzymes have two types of allosteric effector binding sites called the activity site (lacking in certain bacterial ribonucleotide reductases specifically called class Ib) and the specificity site (2). The activity site determines...

Trypanosoma brucei Methylthioadenosine Phosphorylase Protects the Parasite from the Antitrypanosomal Effect of Deoxyadenosine

“…1A). Similarly to what was observed previously (12), the level of ATP, which is needed for the phosphorylation of deoxyadenosine, decreased significantly under these conditions (p Ͻ 0.001, unpaired t test). The level of dCTP increased, and dGTP decreased (Fig.…”

“…A change compared with the previous analyses (12) is that here we used the regular T. brucei HMI-9 medium and did not exclude thymidine. The connection of the HPLC equipment to a flow scintillation analyzer (Radiomatic 150TR, PerkinElmer Life Sciences) enabled the detection of radiolabeled metabolites in experiments where the cells were treated with [2,8-3 H] deoxyadenosine (Moravek Biochemicals, Brea, CA).…”

“…BSFs and Mammalian Cells-Generally, the levels of dNTPs are much lower than NTPs in T. brucei cells (12). However, when cultivated in the presence of 1 mM deoxyadenosine in the growth medium for 1 h, dATP became the most abundant nucleotide in the cell (Fig.…”

“…Nucleotide Pool Measurements-NTP and dNTP pools from T. brucei and mammalian cells were extracted as described previously (12) and quantified by PolyWAX A (PolyLC, Columbia, MD) chromatography (18). A change compared with the previous analyses (12) is that here we used the regular T. brucei HMI-9 medium and did not exclude thymidine.…”

“…As a side activity, T. brucei adenosine kinase is also able to phosphorylate deoxyadenosine (6). This reaction is det-rimental to the parasites because addition of 0.5 mM deoxyadenosine to the culture medium causes the parasites to die within 15 h with grossly elevated dATP levels and a concomitant decrease in ATP, which is consumed in the phosphorylation of the deoxynucleoside (12).…”