CONICET | Buscador de Institutos y Recursos Humanos

Cysteine is essential for the intracellular redox balance and the oxidative detoxification processes. Plants, primitive eukaryotes and prokaryotes, unlike mammals, are able to synthesize de novo cysteine. Two main biosynthetic routes are known, the condensation of O-acetylserine with inorganic sulfur (IS) involving the activity of a cysteine synthase (CS), or alternatively the condensation of serine with homocysteine generating cystathionine which ultimately forms cysteine. The first step of this transsulfuration pathway, like in mammals, depends on the activity of cystathionine beta synthase (CBS). Since very little is known about metabolism of cysteine in TriTryps, L. major CS and CBS were cloned and expressed in E. coli. The biochemical characterization of the recombinant enzymes showed that CS exhibits a specific activity of 140 UE/mg, when O-acetylserine (OAS) and IS were assayed as substrates, a cooperative behavior was observed for both substrates (Hills constants 2.7, 1.6 respectively), We determined affinity constants of 1.5 mM for OAS and 3.6 mM for IS. Like bacterial CSs, the cytosolic leishmanial CS is highly specific for OAS distinguishing from CSs from plants which utilize O-phosphoserine. Cysteine can also be synthesized in a single step by leishmanial CBS which utilizes IS, serine or OAS. However, 4 fold higher activity was obtained when IS and Serine were assayed (13 UE/mg). Leishmanial CBS, like the mammalian CBS, is also to condensate serine and homocysteine (35 UE/mg) to form cystathionine, which might be ultimately converted into cysteine. Spectral analysis showed that Leishmania CBS distinguishes from the mammalian enzymes by the absence of the Hemo binding domain. Our results suggest this pathogen might be able to synthesize cysteine by different routes depending on the sulfur source availability.