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

Abstract

Trichomonas vaginalis is a protozoan parasite of humans that is able to synthesize cysteine de novo using cysteine synthase but does not produce glutathione. In this study, high pressure liquid chromatography analysis confirmed that cysteine is the major intracellular redox buffer by showing that T. vaginalis contains high levels of cysteine (ϳ600 M) comprising more than 70% of the total thiols detected. To investigate possible mechanisms for the regulation of cysteine levels in T. vaginalis, we have characterized enzymes of the mercaptopyruvate pathway. This consists of an aspartate aminotransferase (TvAspAT1), which transaminates cysteine to form 3-mercaptopyruvate (3-MP), and mercaptopyruvate sulfurtransferase (TvMST), which transfers the sulfur of 3-MP to a nucleophilic acceptor, generating pyruvate. TvMST has high activity with 3-MP as a sulfur donor and can use several thiol compounds as sulfur acceptor substrates. Our analysis indicated that TvMST has a k cat /K m for reduced thioredoxin of 6.2 ؋ 10 7 M ؊1 s ؊1 , more than 100-fold higher than that observed for ␤-mercaptoethanol and cysteine, suggesting that thioredoxin is a preferred substrate for TvMST. Thiol trapping and mass spectrometry provided direct evidence for the formation of thioredoxin persulfide as a product of this reaction. The thioredoxin persulfide could serve a biological function such as the transfer of the persulfide to a target protein or the sequestered release of sulfide for biosynthesis. Changes in MST activity of T. vaginalis in response to variation in the supply of exogenous cysteine are suggestive of a role for the mercaptopyruvate pathway in the removal of excess intracellular cysteine, redox homeostasis, and antioxidant defense.

Characterization and Interaction Studies of Two Isoforms of the Dual Localized 3-Mercaptopyruvate Sulfurtransferase TUM1 from Humans

“…The TUM1-bound persulfide can be transferred to acceptors such as cyanide to generate the less toxic thiocyanate (44). Alternatively, H 2 S can be released from TUM1 in the presence of reducing systems like thioredoxin or glutathione (45,46). Inhibition of TUM1 conserves cysteine and contributes to an increase in the cysteine pool (47,48).…”

Novel Trichomonacidal Spermicides

“…Although these cells do not essentially require oxygen for energy metabolism, they are exposed to oxygen in their natural environment inside the host. T. vaginalis lacks glutathione (the intracellular redox buffer), glutathione-dependent peroxidase, and catalase, and therefore it relies heavily on cysteine for protection against oxidative stress, which constitute Ͼ70% of cell's total thiol pool (42). The thioredoxin/thioredoxin-reductase system maintains the thiol status of Trichomonas for deactivating ROS (26).…”

“…The strongly nucleophilic character of the sulfur atom and the unique redox properties of the thiol group make it a key residue for enzyme catalysis, protein folding, and redox signaling and regulation (42), which are important for the cellular energy metabolism, motility, and subsistence of sperm and Trichomonas. We hypothesized that a topical agent capable of targeting free thiols would arrest both sperm and Trichomonas in semen quite specifically since free thiols are unlikely to be available elsewhere in normal vaginal environment due to the low pH.…”

Redox Biochemistry of Hydrogen Sulfide

“…1b. Although the natural sulfide acceptor(s) for MST is not known, like rhodanese (32), MST can transfer the sulfide group to thioredoxin (33).…”