The Crystal Structure of Leishmania major 3-Mercaptopyruvate Sulfurtransferase

Abstract

Leishmania major 3-mercaptopyruvate sulfurtransferase is a crescent-shaped molecule comprising three domains. The N-terminal and central domains are similar to the thiosulfate sulfurtransferase rhodanese and create the active site containing a persulfurated catalytic cysteine (Cys-253) and an inhibitory sulfite coordinated by Arg-74 and Arg-185. A serine protease-like triad, comprising Asp-61, His-75, and Ser-255, is near Cys-253 and represents a conserved feature that distinguishes 3-mercaptopyruvate sulfurtransferases from thiosulfate sulfurtransferases. During catalysis, Ser-255 may polarize the carbonyl group of 3-mercaptopyruvate to assist thiophilic attack, whereas Arg-74 and Arg-185 bind the carboxylate group. The enzyme hydrolyzes benzoyl-Arg-p-nitroanilide, an activity that is sensitive to the presence of the serine protease inhibitor N ␣ -p-tosyl-L-lysine chloromethyl ketone, which also lowers 3-mercaptopyruvate sulfurtransferase activity, presumably by interference with the contribution of Ser-255. The L. major 3-mercaptopyruvate sulfurtransferase is unusual with an 80-amino acid C-terminal domain, bearing remarkable structural similarity to the FK506-binding protein class of peptidylprolyl cis/trans-isomerase. This domain may be involved in mediating protein folding and sulfurtransferase-protein interactions.Sulfurtransferases (EC 2.8.1.1-5) catalyze the transfer of sulfane sulfur from a donor molecule to a thiophilic acceptor. These enzymes are widely distributed in plants, animals, and bacteria (1-3) and have been implicated in a wide range of biological processes. For example, sulfurtransferases may be involved in the formation and maintenance of iron-sulfur clusters in protein (4, 5), detoxification of cyanide (6, 7), degradation of cysteine (8), biosynthesis of the molybdopterin cofactor of xanthine oxidase (9), selenium metabolism (2, 10), and thiamine and 4-thiouridine biosynthesis (11,12). The expression of specific sulfurtransferases is up-regulated under conditions of peroxide or hypo-sulfur stress, osmotic shock, and phage infection (13), suggesting that such enzyme activity is protective of the cell and/or involved in repair processes. Nevertheless, despite intensive study, the biological functions and identification of the physiological substrates of sulfurtransferases remain uncertain.The archetypal sulfurtransferase is rhodanese, a thiosulfate: cyanide sulfurtransferase (TST) 1 able to catalyze the transfer of the thiosulfate sulfur to cyanide in vitro. The related 3-mercaptopyruvate sulfurtransferase (3-mercaptopyruvate:cyanide sulfurtransferase (MST)), first discovered in rat liver (14), catalyzes reactions similar to those catalyzed by rhodanese, but uses 3-mercaptopyruvate in preference to thiosulfate as the donor in the two-step reaction,where E represents the free enzyme and ES the enzyme-sulfur adduct.Crystal structures of rhodaneses have been elucidated and analyzed in detail (15)(16)(17)(18)(19)(20). The enzyme consists of two domains that, despite a low level of sequence iden...

Functional Diversity of the Rhodanese Homology Domain

“…This sequence may be considered a signature for rhodanese domains that use the larger selenophosphate as substrate. The nature of the active site loop of the rhodanese homology domain is important for substrate specificity (12,35). A second feature present in the rhodanese domain of all recog- 4 Whereas our preparations of 2-selenouridine synthase were purified in part using affinity chromatography resulting in Ͼ95% pure protein, biosynthesis of mnm 5 se 2 U from S. typhimurium has been reported to require multiple proteins (47).…”

Production and Physiological Effects of Hydrogen Sulfide

“…Although the total activity of 3MST is greater in the cytosol compared with that in mitochondria, the specific activity is several-fold greater in mitochondria than in the cytosol. 3MST is highly homologous (60% amino acid identity) to rhodanese, which is predominantly localized in mitochondria and has a similar functional structure (4,54,56). The majority of mitochondrial proteins have a signal sequence at their amino terminus that targets these proteins to the mitochondria.…”

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

“…The active site motif of the T. vaginalis protein has an MST consensus (residues 244 -249), differing only in the conservative substitution of isoleucine for valine. A serine peptidase-like triad (His, Ser, and Asp), identified in L. major MST (30), is a common feature of the MST family that distinguishes it from the thiosulfate sulfurtransferase enzymes. His-75 and Ser-255 of L. major MST are conserved in the T. vaginalis enzyme (His-71 and Ser-246), and although Asp-61 of L. major MST is replaced by His-54 in TvMST, the catalytic triad could be completed by an adjacent aspartate (Asp-56).…”