Trypanothione-dependent synthesis of deoxyribonucleotides by Trypanosoma brucei ribonucleotide reductase

Trypanosoma brucei, the causative agent of African sleeping sickness, synth esizes deoxyribonucleotides via a classical eukaryotic class I ribonucleoti de reductase, The unique thiol metabolism of trypanosomatids in which the n early ubiquitous glutathione reductase is replaced by a trypanothione reduc tase prompted us to study the nature of thiols providing reducing equivalen ts for the parasite synthesis of DNA precursors. Here we show that the dith iol trypanothione (bis(glutathionyl)spermidine), in contrast to glutathione , is a direct reductant of T. brucei ribonucleotide reductase with a K-m va lue of 2 mM, This is the first example of a natural low molecular mass thio l directly delivering reducing equivalents for ribonucleotide reduction. At submillimolar concentrations, the reaction is strongly accelerated by tryp aredoxin, a 16-kDa parasite protein with a WCPPC active site motif, The K-m value of T, brucei ribonucleotide reductase for T. brucei tryparedoxin is about 4 muM. The disulfide form of trypanothione is a powerful inhibitor of the tryparedoxin-mediated reaction that may represent a physiological regu lation of deoxyribonucleotide synthesis by the redox state of the cell. The trypanothione/tryparedoxin system is a new system providing electrons for a class I ribonucleotide reductase, in addition to the well known thioredox in and glutaredoxin systems described in other organisms.