Biosynthesis of a Central Intermediate in Hydrogen Sulfide Metabolism by a Novel Human Sulfurtransferase and Its Yeast Ortholog

Abstract

Human sulfide:quinone oxidoreductase (SQOR) catalyzes the conversion of H2S to thiosulfate, the first step in mammalian H2S metabolism. SQOR’s inability to produce the glutathione persulfide (GSS–) substrate for sulfur dioxygenase (SDO) suggested that a thiosulfate:glutathione sulfurtransferase (TST) was required to provide the missing link between the SQOR and SDO reactions. Although TST could be purified from yeast, attempts to isolate the mammalian enzyme were not successful. We used bioinformatic approaches to identify genes likely to encode human TST (TSTD1) and its yeast ortholog (RDL1). Recombinant TSTD1 and RDL1 catalyze a predicted thiosulfate-dependent conversion of glutathione to GSS–. Both enzymes contain a rhodanese homology domain and a single catalytically essential cysteine, which is converted to cysteine persulfide upon reaction with thiosulfate. GSS– is a potent inhibitor of TSTD1 and RDL1, as judged by initial rate accelerations and ≥25-fold lower Km values for glutathione observed in the presence of SDO. The combined action of GSS– and SDO is likely to regulate the biosynthesis of the reactive metabolite. SDO drives to completion p-toluenethiosulfonate:glutathione sulfurtransferase reactions catalyzed by TSTD1 and RDL1. The thermodynamic coupling of the irreversible SDO and reversible TST reactions provides a model for the physiologically relevant reaction with thiosulfate as the sulfane donor. The discovery of bacterial Rosetta Stone proteins that comprise fusions of SDO and TSTD1 provides phylogenetic evidence of the association of these enzymes. The presence of adjacent bacterial genes encoding SDO–TSTD1 fusion proteins and human-like SQORs suggests these prokaryotes and mammals exhibit strikingly similar pathways for H2S metabolism.

Thiosulfate sulfurtransferase-like domain–containing 1 protein interacts with thioredoxin

“…The recombinant protein was purified as described previously (22) and obtained in a yield of 20 mg/liter of culture. TSTD1 was judged to be >95% pure by SDS-PAGE analysis (Fig.…”

“…Cells were harvested by centrifugation at 10,800 × g for 20 min at 4 °C. TSTD1 was purified as described previously with the following modifications (22). The cell pellet from a 6-liter culture was resuspended in 150 mL of 50 mM Tris buffer, pH 8, containing 0.15 M NaCl (Buffer A), 1 tablet of protease inhibitor cocktail (Roche), DNase (3 mg), soybean trypsin inhibitor (2 mg), aprotinin (3 mg), phenylmethanesulfonyl fluoride (4 mg), tosyllysine chloromethyl ketone (0.45 mg) and MgSO 4 (5 mM final concentration) were added to the cell suspension and stirred at 4 °C for 60 min followed by sonication on ice with the following pulse sequence: 30 sec burst, 1 min rest for a total burst time of 10 min at a power output setting of 6.…”

“…At least three variations of the rhodanese domain are found in family members (20). In some members, only a single rhodanese domain is present such as in the bacterial GlpE (21) and human TSTD1 (22). Alternatively, the rhodanese domain can be present in a tandem repeat, as in rhodanese and MST, where the active sites are located in a cleft between the N-and C-terminal domains (14,15).…”

“…Recombinant human TSTD1 utilizes thiosulfate as a sulfur donor, although the kinetics of this reaction were only characterized at the alkaline pH of 9.2 (22). Based on this activity, the involvement of TSTD1 in the sulfide oxidation pathway was proposed, although the remaining enzymes in this pathway exist inside the mitochondrion.…”

Polymorphic Variants of Human Rhodanese Exhibit Differences in Thermal Stability and Sulfur Transfer Kinetics

“…It has also been proposed that the role of rhodanese or another thiol sulfurtransferase in the mitochondrial sulfide oxidation pathway is to utilize thiosulfate, forming GSSH instead ( Fig. 1E) (12,13). However, the efficiency of thiosulfate production by wildtype rhodanese is much greater than that for thiosulfate utili-zation (11).…”