Characterization of transsulfuration and cysteine biosynthetic pathways inthe protozoan hemoflagellate, Trypanosoma cruzi - Isolation and molecular characterization of cystathionine beta-synthase and serine acetyltransferase from Trypanosoma

Sulfur-containing amino acids play an important role in a variety of cellul ar functions such as protein synthesis, methylation, and polyamine and glut athione synthesis. We cloned and characterized cDNA encoding cystathionine beta -synthase (CBS), which is a key enzyme of transsulfuration pathway, fr om a hemoflagellate protozoan parasite Trypanosoma cruzi. T. cruzi CBS, unl ike mammalian CBS, lacks the regulatory carboxyl terminus, does not contain heme, and is not activated by S-adenosylmethionine. T. cruzi CBS mRNA is e xpressed as at least six independent isotypes with sequence microheterogene ity from tandemly linked multicopy genes. The enzyme forms a homotetramer a nd, in addition to CBS activity, the enzyme has serine sulfhydrylase and cy steine synthase (CS) activities in vitro. Expression of the T. cruzi CBS in Saccharomyces cerevisiae and Escherichia coli demonstrates that the CBS an d CS activities are functional in vivo. Enzymatic studies on T. cruzi extra cts indicate that there is an additional CS enzyme and stage-specific contr ol of CBS and CS expression. We also cloned and characterized cDNA encoding serine acetyltransferase (SAT), a key enzyme in the sulfate assimilatory c ysteine biosynthetic pathway. Dissimilar to bacterial and plant SAT, a reco mbinant T. cruzi SAT showed allosteric inhibition by L-cysteine, L-cystine, and, to a lesser extent, glutathione. Together, these studies demonstrate the T. cruzi is a unique protist in possessing both transsulfuration and su lfur assimilatory pathways.