Cysteine Biosynthesis in Trichomonas vaginalis Involves Cysteine Synthase Utilizing O-Phosphoserine

Abstract

Trichomonas vaginalis is an early divergent eukaryote with many unusual biochemical features. It is an anaerobic protozoan parasite of humans that is thought to rely heavily on cysteine as a major redox buffer, because it lacks glutathione. We report here that for synthesis of cysteine from sulfide, T. vaginalis relies upon cysteine synthase. The enzyme (TvCS1) can use either O-acetylserine or O-phosphoserine as substrates. The K m values of the enzyme for sulfide are very low (0.02 mM), suggesting that the enzyme may be a means of ensuring that sulfide in the parasite is maintained at a low level. T. vaginalis appears to lack serine acetyltransferase, the source of O-acetylserine in many cells, but has a functional 3-phosphoglycerate dehydrogenase and an O-phosphoserine aminotransferase that together result in the production of O-phosphoserine, suggesting that this is the physiological substrate. TvCS1 can also use thiosulfate as substrate. Overall, TvCS1 has substrate specificities similar to those reported for cysteine synthases of Aeropyrum pernix and Escherichia coli, and this is reflected by sequence similarities around the active site. We suggest that these enzymes are classified together as type B cysteine synthases, and we hypothesize that the use of O-phosphoserine is a common characteristic of these cysteine synthases. The level of cysteine synthase in T. vaginalis is regulated according to need, such that parasites growing in an environment rich in cysteine have low activity, whereas exposure to propargylglycine results in elevated cysteine synthase activity. Humans lack cysteine synthase; therefore, this parasite enzyme could be an exploitable drug target.

The Mercaptopyruvate Sulfurtransferase of Trichomonas vaginalis Links Cysteine Catabolism to the Production of Thioredoxin Persulfide

“…Cloning and Expression-T. vaginalis genomic DNA was prepared as described previously (10). The putative MST gene, TVAG_129830, was amplified from genomic DNA using primers NT386 (5Ј-CAGCCATGGTTGAGACAATTGTAA-CACC-3Ј) and NT387 (5Ј-CACTCGAGTTCCTGGTATG-GATTTGGTCC-3Ј) and Hi-Fidelity polymerase under the following conditions: 94°C for 2 min; 25 cycles of 94°C for 15 s, 55°C for 30 s, and 72°C for 1 min; and then a further 7 min at 72°C.…”

“…Cysteine itself is maintained at relatively low levels, sufficient for protein synthesis and production of essential metabolites but below the threshold of toxicity (14). T. vaginalis does not synthesize glutathione, and cysteine is believed to be the main intracellular antioxidant in this organism (9,10,15). This raises questions as to how T. vaginalis regulates intracellular cysteine levels so that it functions effectively as a redox buffer without inducing a toxic effect on the cell.…”

“…MST gene (TVAG_129830) by blastP search of the T. vaginalis genome data base was reported previously (10). The 879-bp reading frame encodes a 292-residue protein with a predicted size of 32.7 kDa.…”

“…vaginalis appears to be able to obtain cysteine from its environment but also has the ability to synthesize cysteine de novo from phosphoserine and sulfide with cysteine synthase (TvCS) 2 (Ref. 10 and Fig. 1).…”

“…Growth of T. vaginalis-T. vaginalis G3 was grown in 25 ml of modified Diamond's medium with little gas phase (10). The cultures were initiated with 10 5 cells ml Ϫ1 and harvested by centrifugation after 18 h of growth at 37°C.…”