THE STRUCTURE, ORGANIZATION, AND EXPRESSION OF THE LEISHMANIA-DONOVANI GENE ENCODING TRYPANOTHIONE REDUCTASE

Trypanothione reductase (TR) is an NADPH-dependent flavoprotein oxidor eductase central to thiol metabolism in the trypanosomatids. We report here the cloning by expression of the Leishmania donovani gene. It is single copy, expresses a 2.6-kb transcript and a 52-kDa protein and i s located on a 1.1-Mbp chromosome. The 491 amino acid sequence has 76% similarity to Crithidia fasciculata and 67% similarity to Trypanosoma cruzi, Trypanosoma congolense and Trypanosoma brucei TR. Residues rec ognising the adenosine pyrophosphate moiety of NADPH and FAD, and resi dues in the catalytic site segment (A(47)-A(67)) involving electron tr ansfer from TR to trypanothione disulphide (T(S)(2)) were completely c onserved. Thus inhibitors of TR are likely to be active against the en zyme from all the parasitic trypanosomatids. Two peptide inserts (39-4 7, 131-140) seen in other TR genes and a C-terminal extension of 19 re sidues were also present. When the gene was introduced back into L. do novani at high copy number using the pTEX expression vector, we detect ed elevated expression of TR RNA and a 13-fold increase in TR activity . Transfection and overexplession of the TR gene will facilitate studi es of gene function and of the dependence of trypanosomatids on TR for protection against oxidative stress.