CLONING, SEQUENCING AND FUNCTIONAL EXPRESSION OF DIHYDROLIPOAMIDE DEHYDROGENASE FROM THE HUMAN PATHOGEN TRYPANOSOMA-CRUZI

This work presents the complete sequences of a cDNA and the two alleli c genes of dihydrolipoamide dehydrogenase (LipDH) from Trypanosoma cru zi, the causative agent of Chagas' disease (American trypanosomiasis). The full-length cDNA has an ORF of 1431 bp and encodes a protein of 4 77 amino acid residues. LipDH is a homodimeric protein with FAD as pro sthetic group. The calculated molecular mass of the subunit of the mat ure protein with bound FAD is 50066. Comparison of the deduced amino a cid sequence of LipDH from T. cruzi with that of Trypanosoma brucei an d man shows identities of 81% and 50%, respectively. An N-terminal non apeptide, not present in the mature enzyme, represents a mitochondrial targeting sequence so far found only in trypanosomatids. The gene lpd l of T. cruzi LipDH was expressed without the targeting sequence in Es cherichia coli JRG1342 cells which are deficient for LipDH. For this p urpose an ATG codon was introduced directly upstream the codon for Asn 10 which represents the N-terminus of the mature protein. This system allowed the synthesis of 1000 U T. cruzi LipDH/l bacterial cell cultur e. The recombinant protein was purified to homogeneity by (NH4)(2)SO4- precipitation and affinity chromatography on 5' AMP-Sepharose. The K-m values for NAD(+), NADH, lipoamide and dihydrolipoamide are identical with those of the enzyme isolated from the parasite. LipDH is present in all major developmental stages of T. cruzi as shown by northern an d western blot analyses. This finding is in agreement with the citric acid cycle being active throughout the whole life cycle of the parasit e. In vitro studies on a mammalian LipDH revealed the ability of the f lavoenzyme to catalyze the redoxcycling and superoxide anion productio n of nitrofuran derivatives including the antitrypanosomal drug Nifurt imox. For that reason T. cruzi LipDH is regarded as a promising target for the structure-based development of new antiparasitic drugs. The b acterial expression system for the parasite enzyme will now allow the study of the role of T. cruzi LipDH in drug activation and the crystal lization of the protein.