CLONING AND CHARACTERIZATION OF THE R1 AND R2 SUBUNITS OF RIBONUCLEOTIDE REDUCTASE FROM TRYPANOSOMA-BRUCEI

Ribonucleotide reductase (RNR) catalyzes the rate limiting step in the de novo synthesis of deoxyribonucleotides by directly reducing ribonu cleotides to the corresponding deoxyribonucleotides. To keep balanced pools of deoxyribonucleotides, all nonviral RNRs studied so far are al losterically regulated, Most eukaryotes contain a class I RNR, which i s a heterodimer of two nonidentical subunits called proteins R1 and R2 , We have isolated cDNAs encoding the R1 and R2 proteins from Trypanos oma brucei. The amino acid sequence identities with the mouse R1 and R 2 subunits are 58% and 63%, respectively, Recombinant active trypanoso me 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'-diph osphate reduction by the trypanosome RNR in the presence of various al losteric effecters showed that the activity is highest with dTTP, dGTP , or dATP and considerably lower with ATP. The effect of dGTP is eithe r activating (alone) or inhibitory (in the presence of ATP), Filter bi nding studies indicated that there are two classes of allosteric effec tor binding sites that bind ATP or dATP (low-affinity dATP site) and A TP, dATP, dGTP, or dTTP (high-affinity dATP site), respectively, There fore, the structural organization of the allosteric sites is very simi lar to the mammalian RNRs, whereas the allosteric regulation of cytidi ne 5'-diphosphate reduction is unique, Hopefully, this difference can be used to target the trypanosome RNR for therapeutic purposes.