Characterization of Transsulfuration and Cysteine Biosynthetic Pathways in the Protozoan Hemoflagellate, Trypanosoma cruzi

Abstract

Sulfur-containing amino acids play an important role in a variety of cellular functions such as protein synthesis, methylation, and polyamine and glutathione synthesis. We cloned and characterized cDNA encoding cystathionine ␤-synthase (CBS), which is a key enzyme of transsulfuration pathway, from a hemoflagellate protozoan parasite Trypanosoma cruzi. T. cruzi CBS, unlike mammalian CBS, lacks the regulatory carboxyl terminus, does not contain heme, and is not activated by S-adenosylmethionine. T. cruzi CBS mRNA is expressed as at least six independent isotypes with sequence microheterogeneity from tandemly linked multicopy genes. The enzyme forms a homotetramer and, in addition to CBS activity, the enzyme has serine sulfhydrylase and cysteine synthase (CS) activities in vitro. Expression of the T. cruzi CBS in Saccharomyces cerevisiae and Escherichia coli demonstrates that the CBS and CS activities are functional in vivo. Enzymatic studies on T. cruzi extracts indicate that there is an additional CS enzyme and stage-specific control of CBS and CS expression. We also cloned and characterized cDNA encoding serine acetyltransferase (SAT), a key enzyme in the sulfate assimilatory cysteine biosynthetic pathway. Dissimilar to bacterial and plant SAT, a recombinant 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 sulfur assimilatory pathways.Various sulfur compounds, especially cysteine, methionine, and S-adenosylmethionine, are essential for the growth and activities of all cells (1, 2). Methionine initiates the synthesis of proteins, whereas cysteine plays a critical role in the structure, stability, and catalytic function of many proteins. S-Adenosylmethionine plays a crucial role in methyl group transfer and in polyamine biosynthesis. Cysteine is also involved in the synthesis of the major antioxidant glutathione.In the filamentous fungi, Aspergillus nidulans and Neurospora crassa, the major route for the synthesis of cysteine is the condensation of O-acetylserine (OAS) 1 with sulfide, catalyzed by cysteine synthase (CS, OAS sulfhydrase) (3, 4). This pathway has been shown to be present in prokaryotes, plants, and enteric protozoan Entamoebae, and is generally called assimilatory cysteine biosynthetic pathway since this process involves reduction and fixation of inorganic sulfate to organic amino acids. Cysteine can also be synthesized by an alternative pathway: the sulfurylation of O-acetylhomoserine to give homocysteine, which then can be converted to cysteine via cystathionine by the transsulfuration pathway. In vertebrates, cysteine is synthesized from methionine via cystathionine by the transsulfuration pathway. This pathway is believed to be the sole route for cysteine synthesis in vertebrates with cystathionine ␤-synthase (CBS) acting as the flux-controlling enzyme (1). In mammals, this pathway also functions as catabolic pathway of methionine a...

Deletion Mutagenesis of Human Cystathionine β-Synthase

“…Recently, work from another group has suggested that the formation of CBS tetramers may involve residues outside of the C-terminal domain (20). The series of deletion mutants generated in this present study provides us with a useful tool to further delineate the one or more regions of CBS associated with the formation of tetramers.…”

“…The function of this ligand is unknown, but a number of studies indicate it may play either a regulatory or structural role (13)(14)(15)(16). The observation that both yeast (17)(18)(19) and Trypanosoma cruzi (20) CBS lack heme indicates that it is not directly involved in catalysis (18,19).…”

“…CBS from T. cruzi, which is also unresponsive to AdoMet, lacks the typical CBS C-terminal region and exists predominantly as a tetramer. This observation has lead to speculation that CBS tetramerization is not exclusively a function of the C-terminal region (20).…”

3-Mercaptopyruvate Sulfurtransferase of LeishmaniaContains an Unusual C-terminal Extension and Is Involved in Thioredoxin and Antioxidant Metabolism

“…We have shown that LmajMST works in conjunction with both recombinant L. major cysteine synthase (GenBank TM accession number AL499624) and recombinant L. major cystathionine ␤-synthase (accession number AL499619) to synthesize cysteine in vitro. T. cruzi cystathionine ␤-synthase was also recently reported to possess cysteine synthase activity (45).…”

Purification and characterization of the wild type and truncated human cystathionine β-synthase enzymes expressed in E. coli

“…An evolutionary relationship between these enzymes is also illustrated by the fact that some CBS enzymes have retained CS activity [4]. CS and CBS, together with other enzymes such as β-cyanoalanine synthase, the β-subunit of tryptophan synthase and cysteine lyase, catalyze primarily β-replacement reactions [5,6].…”