Cloning and characterization of the R1 and R2 subunits of ribonucleotide reductase from Trypanosoma brucei

Abstract

Ribonucleotide reductase (RNR) catalyzes the rate limiting step in the de novo synthesis of deoxyribonucleotides by directly reducing ribonucleotides to the corresponding deoxyribonucleotides. To keep balanced pools of deoxyribonucleotides, all nonviral RNRs studied so far are allosterically regulated. Most eukaryotes contain a class I RNR, which is a heterodimer of two nonidentical subunits called proteins R1 and R2. We have isolated cDNAs encoding the R1 and R2 proteins from Trypanosoma brucei. The amino acid sequence identities with the mouse R1 and R2 subunits are 58% and 63%, respectively. Recombinant active trypanosome R1 and R2 proteins were expressed in Escherichia coli and purified. The R2 protein contains an iron-tyrosyl free radical center verified by EPR spectroscopy and iron analyses. Measurement of cytidine 5 -diphosphate reduction by the trypanosome RNR in the presence of various allosteric effectors showed that the activity is highest with dTTP, dGTP, or dATP and considerably lower with ATP. The effect of dGTP is either activating (alone) or inhibitory (in the presence of ATP). Filter binding studies indicated that there are two classes of allosteric effector binding sites that bind ATP or dATP (low-affinity dATP site) and ATP, dATP, dGTP, or dTTP (high-affinity dATP site), respectively. Therefore, the structural organization of the allosteric sites is very similar to the mammalian RNRs, whereas the allosteric regulation of cytidine 5 -diphosphate reduction is unique. Hopefully, this difference can be used to target the trypanosome RNR for therapeutic purposes.Sleeping sickness is a major health problem in a predominant part of sub-Saharan Africa. The disease, caused by a unicellular eukaryote called Trypanosoma brucei (1), is usually fatal if left untreated, and Ϸ25,000 new cases are diagnosed each year. However, since only a small fraction of the population in high risk areas is under surveillance, the World Health Organization has estimated the real figure to be Ϸ300,000. ‡ The current chemotherapy of sleeping sickness suffers from various limitations. Many of the compounds used are highly toxic and there is also a widespread drug resistance among the trypanosomes (2, 3).Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides. There are three different classes of RNRs (4). The substrate, which is a nucleoside diphosphate for the class I enzymes, is reduced through a radical mechanism. Class I RNR is a heterodimeric enzyme of ␣ 2 ␤ 2 type. The large R1 subunit binds substrates and allosteric effectors, while the small R2 subunit contains a tyrosyl radical (5), generated by a binuclear ferric iron center. The tyrosyl radical in the R2 protein is linked to the active site in the R1 subunit through a hydrogen-bonded long-range electron transport chain (6-10). Class II RNR generates a radical from 5Ј-deoxyadenosylcobalamin (11, 12) and class III RNR, which only works under anaerobic conditions, contains a stable glycyl radical (13). Class I and to s...

The Dithiol Glutaredoxins of African Trypanosomes Have Distinct Roles and Are Closely Linked to the Unique Trypanothione Metabolism

“…T(SH) 2 (27), T. brucei Tpx (19), T. cruzi TR (28), T. brucei glutathione peroxidase type tryparedoxin peroxidase (Px III) (29), T. brucei His 6 -peroxiredoxin type tryparedoxin peroxidase (TXNPx) (30), the small subunit of T. brucei RR (R2) (31), and His-tagged E. coli cysteine desulfurase (9) were prepared as described. The gene for the large subunit (R1) of RR was cloned in the modified pET vector described below, and the tag-free protein was purified by chromatography on two consecutive nickel-NT-Sepharose columns as outlined below for Grx1 and Grx2.…”

Identification and Functional Characterization of Thioredoxin from Trypanosoma brucei brucei

“…Anders Hofer and Lars Thelander, Umeå, Sweden. Recombinant T. brucei tryparedoxin (8), T. cruzi trypanothione reductase (10,11), and T. brucei ribonucleotide reductase (12)(13)(14) were prepared as described. Polyclonal rabbit antibodies against the recombinant T. brucei thioredoxin were produced by Eurogentec.…”

Cloning, sequencing and expression of ribonucleotide reductase R2 from Trypanosoma brucei

“…Note: Recently the sequences of the RI and R2 subunits of T. brucei ribonucleotide reductase have been published [29]. There are two discrepancies between the deduced protein sequences of the R2 protein.…”

“…There are two discrepancies between the deduced protein sequences of the R2 protein. Arg8 and Lys43 (this paper) correspond to Cys8 and Glu43 [29], respectively. Both differences represent single base exchanges.…”