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. 2009 Nov 27;284(48):33485-94.
doi: 10.1074/jbc.M109.054320. Epub 2009 Sep 17.

V体育平台登录 - The mercaptopyruvate sulfurtransferase of Trichomonas vaginalis links cysteine catabolism to the production of thioredoxin persulfide

Gareth D Westrop  1 , Ina Georg, Graham H Coombs
Affiliations

The mercaptopyruvate sulfurtransferase of Trichomonas vaginalis links cysteine catabolism to the production of thioredoxin persulfide

V体育官网入口 - Gareth D Westrop et al. J Biol Chem. .

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 ( approximately 600 mum) 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 x 10(7) m(-1) s(-1), more than 100-fold higher than that observed for beta-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 VSports手机版. 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. .

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Figures

FIGURE 1.
FIGURE 1.
Sulfur amino acid metabolism in T. vaginalis. MGL, methionine γ-lyase; MAT, methionine adenosyltransferase; SAM-MT, S-adenosylmethionine-dependent methyltransferases; SAHH, S-adenosylhomocysteine hydrolase; PGDH, phosphoglycerate dehydrogenase; PSAT, phosphoserine aminotransferase; CD, cysteine desulfurase; R-SH, thiol compound; R-S-SH, persulfide compound.
FIGURE 2.
FIGURE 2.
Recombinant proteins used in activity analyses. Lane 1, 5 μg of rTvAspAT1; lane 2, 5 μg of rTvMST.
FIGURE 3.
FIGURE 3.
Proposed mechanisms of the reactions catalyzed by MST. a, MST activity. Part i, MST catalyzes the transfer of sulfur from 3-MP to a nucleophilic acceptor such as a thiol (R-SH), generating activated (sulfane) sulfur in the form of a persulfide (R-S-SH). Part ii, in the presence of an excess of thiol, the persulfide is reduced spontaneously generating H2S and a disulfide (R-S-S-R). b, thioredoxin oxidation with 3-MP as a sulfur donor. Part i, thioredoxin acts as a sulfur acceptor in the sulfurtransferase reaction catalyzed by MST, and one of the thioredoxin active site cysteines is converted to cysteine persulfide. Part ii, this is followed by a spontaneous reaction in which sulfide is released and thioredoxin is oxidized. c, thioredoxin oxidation without a sulfur donor. Part i, oxidation of active site cysteine forms a sulfenate (-S-OH). Part ii, reduction of sulfenate by thioredoxin regenerates active site cysteine and thioredoxin is oxidized.
FIGURE 4.
FIGURE 4.
Sulfurtransferase activity with reduced thioredoxin as sulfur acceptor, determined by quantification of thioredoxin oxidation. a, change in the rate of NADPH oxidation following the addition of rTvMST and 3-MP to a thioredoxin-reducing system. The initial reaction contained 0.1 μm rTvTrxR, 12.5 μm rTvTrx, and 0.2 mm NADPH. NADPH oxidation was followed by measuring absorbance at 340 nm. rTvMST (E) and 3-MP (S) were added at the times indicated to give final concentrations of 10 nm and 0.3 mm, respectively. ▴, reaction with rTvMST; ○, reaction without rTvMST. b, variation of sulfurtransferase activity of rTvMST with thioredoxin concentration. Inset, double-reciprocal plot. The reactions contained 0.3 mm 3-MP with 0.8–12.5 μm rTvTrx.
FIGURE 5.
FIGURE 5.
Sulfurtransferase activity with reduced thioredoxin as sulfur acceptor, determined by quantification of sulfide production. a, change in the rate of sulfide production following the addition of 1.5 nm rTvMST to a reaction containing 1 mm DTT, 12.5 μm rTVTrx, and 0.3 mm 3-MP. Sulfide production was quantified by measuring the change in absorbance at 360 nm. ▴, reaction with rTvMST; ○, reaction without rTvMST. b, variation of sulfurtransferase activity of rTvMST with thioredoxin concentration. Inset, double-reciprocal plot. The reactions contained 0.3 mm 3-MP with 0.5–15 μm rTvTrx.
FIGURE 6.
FIGURE 6.
Changes in MST enzymatic activity under different conditions. Shown are the MST activities in nmol min−1 mg of protein−1. The values are the means ± S.D. from three replicate assays for each extract. The different growth conditions were: control (lane 1), with added 10 mm cysteine (lane 2), and with 5 μm propargylglycine (lane 3).
FIGURE 7.
FIGURE 7.
Thiol content of T. vaginalis grown under different conditions. a, cysteine (nmol (108 cells)−1). b, homocysteine (nmol (108 cells)−1). The results are the means ± S.D. from three extracts. The different growth conditions were: control (lane 1), with added 10 mm cysteine (lane 2), and with 5 μm propargylglycine (lane 3).
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