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Abstract
Trypanosoma brucei, the causative agent of African sleeping sickness, synthesizes deoxyribonucleotides via a classical eukaryotic class I ribonucleotide reductase. The unique thiol metabolism of trypanosomatids in which the nearly ubiquitous glutathione reductase is replaced by a trypanothione reductase prompted us to study the nature of thiols providing reducing equivalents for the parasite synthesis of DNA precursors. Here we show that the dithiol trypanothione (bis(glutathionyl)spermidine), in contrast to glutathione, is a direct reductant of T. brucei ribonucleotide reductase with a K(m) value of 2 mm. This is the first example of a natural low molecular mass thiol directly delivering reducing equivalents for ribonucleotide reduction. At submillimolar concentrations, the reaction is strongly accelerated by tryparedoxin, 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 micrometer. The disulfide form of trypanothione is a powerful inhibitor of the tryparedoxin-mediated reaction that may represent a physiological regulation 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 thioredoxin and glutaredoxin systems described in other organisms.
Highlights
R1 protein harbors the active site, as well as regulatory sites, and the small R2 protein contains a -oxo-bridged diiron cluster, which represents the Fe(III)–O2Ϫ–Fe(III) cofactor in all eukaryotic ribonucleotide reductases, and a tyrosyl radical essential for catalysis [1, 5]
We will show that the trypanosomatid-specific dithiol trypanothione, in contrast to the monothiol glutathione, is a direct donor of reducing equivalents for T. brucei ribonucleotide reductase and that the reaction is catalyzed by tryparedoxin
Different Thiols as Hydrogen Donors of T. brucei Ribonucleotide Reductase—Formation of [3H]dGDP from [3H]GDP by T. brucei ribonucleotide reductase was followed in the presence of trypanothione, glutathionylspermidine, glutathione, and the nonphysiological dithiol DTE
Summary
R1 and R2, large and small subunit, respectively, of ribonucleotide reductase; DTE, dithioerythritol; DTNB, 5,5Ј-dithiobis(2-nitrobenzoate); Gsp, (mono)glutathionylspermidine; T(SH), trypanothione [N1,N8-bis(glutathionyl)spermidine]; TS2, trypanothione disulfide (oxidized trypanothione); TR, trypanothione reductase; HPLC, high pressure liquid chromatography. Monoglutathionylspermidine (Gsp) and trypanothione (N1,N8-bis(glutathionyl)spermidine; T(SH)2) are the main low molecular mass thiols and are responsible for the redox balance of the cell [11, 12] These glutathionylspermidine conjugates are kept reduced by the flavoenzyme trypanothione reductase (TS2 ϩ NADPH ϩ Hϩ 3 T(SH) ϩ NADP), an essential enzyme of the parasite [13, 14]. We will show that the trypanosomatid-specific dithiol trypanothione, in contrast to the monothiol glutathione, is a direct donor of reducing equivalents for T. brucei ribonucleotide reductase and that the reaction is catalyzed by tryparedoxin.
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