Biological and functional studies of enzymes involved in the synthesis of cysteine in Trypanosoma cruziand Leishmania parasites

GIORDANA LUCILA

Montevideo

Simposio; Symposium ?Thiol Metabolism and Redox Regulation of Cellular Functions?; 2015

International Centre for Genetic Engineering and Biotechnology

Particularly in Leishmania spp. and trypanosomes, cysteine is an essential building block for trypanothione biosynthesis. This abundant (in the mM range) low-molecular-mass dithiol maintains the redox homeostasis in all of the developmental stages of these pathogenic protists.Trypanosoma cruzi and Leishmania parasites, unlike humans, seem capable of biosynthesize cysteine de novo. In these pathogens, cysteine synthase (CS) and cystathionine betta-synthase (CBS) are expected to participate in this metabolic process. Moreover, the reverse transulfuration pathway (RTP) is also predicted to be operative; in these parasites CBS catalyzes the production of cystathionine and the latter is cleaved into cysteine by cystathionine gamma-lyase (CGL). CBS, CGL and CS are cytosolic enzymes, notably only CS and CGL are expressed along the whole life cycle of both parasites. CGLs are piridoxal phosphate (PLP) dependent enzymes that are comprised within the subgroup of the Cys-Met metabolism related proteins. Despite of the structure similarity between all the members of this family, they differ in substrate specificity and catalytic capacities. T. cruzi CGL differ from the mammalian homologue given that the former is unable to produce H2S as well as is remarkably more susceptible towards DL-propargylglycine (PAG) a well known inhibitor of CGLs (IC50 12 μM vs 2mM).Even though Leishmania major CGL reveals remarkable sequence identity (> 80%) with the functional T. cruzi homolog, unlike the latter the recombinant leishmanial CGL could not be functionally expressed in Escherichia coli cultures. This fact prompted us to undertake the approach of functional complementation of a Saccharomyces cerevisiae CGL-null mutant to assess the activity of this enzyme in Leishmania parasites. The co-existence ofde novo synthesis with the RTP is extremely rare in most living organisms; however PAG arrests the proliferation of L. major promastigotes (IC50of aprox 65uM).These findings raise questions regarding the biological role of CGL in these pathogens as well as indicate the need of enlarging our knowledge about its structural features in order to disclose the particularities of the active site of this enzyme as well as examine its potentiality as drug target