Structural and biochemical analyses indicate that a bacterial persulfide dioxygenase–rhodanese fusion protein functions in sulfur assimilation

Motl, Nicole; Skiba, Meredith A.; Kabil, Ömer; Smith, Janet L.; Banerjee, Ruma

doi:10.1074/jbc.m117.790170

Cited by 32 publications

(48 citation statements)

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“…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). Finally, rhodanese domains are fused to other protein domains as in Cdc25 phosphatase (20) or in the PRF and CstB proteins (23,24).Sulfurtransferases are ubiquitously distributed enzymes that function in a range of pathways including sulfur metabolism, iron-sulfur cluster formation, selenium metabolism, and cyanide detoxification (13,14,(25)(26)(27)(28). In humans, there are five sulfurtransferases including mitochondrial rhodanese and MST, which is found in the cytoplasmic and mitochondrial compartments (29,30).…”

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

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Motl

Akey

et al. 2018

Journal of Biological Chemistry

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ǂ These authors contributed equally to this work Rhodanese domains are structural modules present in the sulfurtransferase superfamily. These domains can exist as single units, in tandem repeats or fused to domains with other activities. Despite their prevalence across species, the specific physiological roles of most sulfurtransferases are not known. Mammalian rhodanese and mercaptopyruvate sulfurtransferase are perhaps the best-studied members of this protein superfamily and are involved in hydrogen sulfide metabolism. The relatively unstudied human thiosulfate sulfurtransferase like domain-containing 1 (TSTD1) protein, a single-domain cytoplasmic sulfurtransferase, was also postulated to play a role in the sulfide oxidation pathway using thiosulfate to form glutathione persulfide, for subsequent processing in the mitochondrial matrix. Prior kinetic analysis of TSTD1 was performed at pH 9.2, raising questions about relevance and the proposed model for TSTD1 function. In this study, we report a 1.04 Å resolution crystal structure of human TSTD1, which displays an exposed active site that is distinct from that of rhodanese and mercaptopyruvate sulfurtransferase. Kinetic studies with a combination of sulfur donors and acceptors reveal that TSTD1 exhibits a low K M for thioredoxin as a sulfane sulfur acceptor and that it utilizes thiosulfate inefficiently as a sulfur donor. The active site exposure and its interaction with thioredoxin, suggest that TSTD1 might play a role in sulfide-based signaling. The apical localization of TSTD1 in human colonic crypts, which interfaces with sulfide-releasing microbes, and the overexpression of TSTD1 in colon cancer, provides potentially intriguing clues as to its role in sulfide metabolism. Hydrogen sulfide (H 2 S)1 is an important signaling molecule with effects on multiple physiological processes including neuromodulation, inflammation and cardiac function (1-7). Maintaining healthy levels of H 2 S in mammalian cells requires tight control of its biosynthesis and its catabolism (8,9). H 2 S is produced endogenously by two enzymes in the transsulfuration pathway, cystathionine β-synthase and γ-cystathionase as well as by mercaptopyruvate sulfurtransferase (MST) (10-13). H 2 S is oxidized via the sulfide oxidation pathway to generate the end-products thiosulfate Protein persulfidation, a posttranslational modification of cysteine residues, is postulated to be a major mechanism by which H 2 S signals (17). However, the mechanism by which target proteins acquire this modification and the sulfur source(s) used in persulfidation reactions are unclear (18). Sulfurtransferases like rhodanese or the single rhodanese domain containing TSTD1 could potentially play a role in protein persulfidation because of their capacity to form an active site cysteine persulfide during their reaction cycle (18).The rhodanese superfamily members are distinguished by a structural module with an α/β topology in which α-helices surround a central five-stranded β-sheet core (19). At least three variations...

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

Libiad

Motl

Akey

et al. 2018

Journal of Biological Chemistry

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“…The presence of GHcy-SO 2 H as the only detectable product is consistent with the inability of PDO to process GHcySH through the ultimate hydrolysis step. Crystal structures of PDO homologs indicate that only the sulfur atom of the substrate coordinates to the iron, displacing a single water molecule (16,17). This is similar to isopencillin Nsynthase (22) but contrasts with cysteine dioxygenase, in which the substrate serves as a bidentate ligand donating both the sulfur and the α-amino group (20).…”

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confidence: 98%

“…Studies on the Pseudomonas putida PDO and the Burkholderia phytofirmans PRF, a fusion protein containing a PDO domain, have shown that the substrate is within hydrogen bonding distance to the residue corresponding to Arg-163 in human PDO (14,16,17). Arg-163 is involved in hydrogen bonding interactions with Asp-165 and Ser-230 (Fig.…”

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confidence: 99%

“…The active site iron is located inside a large groove that is framed by positively charged residues on one side and a polar tyrosine on the other (14). From the crystal structures of PDO homologs with GSH bound (16,17) and of human PDO with GSSH docked (14), the substrate binding residues have been identified as , and potentially, Arg-214, which is predicted to be within hydrogen bonding distance to GSSH.While the mechanisms of several members of the mononuclear non-heme iron dioxygenases have been studied in detail (18,19), the mechanism of PDO has not been investigated. Based on the general mechanism of non-heme iron dioxygenases and, more specifically, cysteine dioxygenase (20), a mechanism was proposed for PDO ( Fig.…”

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Mechanism-based inhibition of human persulfide dioxygenase by γ-glutamyl-homocysteinyl-glycine

Kabil

Motl

Strack

et al. 2018

Journal of Biological Chemistry

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Hydrogen sulfide (H 2 S) is a signaling molecule with many beneficial effects. However, its cellular concentration is strictly regulated to avoid toxicity. Persulfide dioxygenase (PDO or ETHE1) is a mononuclear non-heme ironcontaining protein in the sulfide oxidation pathway catalyzing the conversion of glutathione persulfide (GSSH) to sulfite and glutathione. PDO mutations result in the autosomal recessive disorder, ethylmalonic encephalopathy (EE). Here, we developed γ-glutamyl-homocysteinyl-glycine (GHcySH) in which the cysteinyl moiety in glutathione is substituted with homocysteine, as a mechanismbased PDO inhibitor. Human PDO used GHcySH as an alternate substrate and converted it to GHcy-SO 2 H, mimicking GS-SO 2 H, the putative oxygenated intermediate formed with the natural substrate. Since GHcy-SO 2 H contains a C-S bond rather than an S-S bond in GS-SO 2 H, it failed to undergo the final hydrolysis step in the catalytic cycle, leading to PDO inhibition. We also characterized the biochemical penalties incurred by the L55P, T136A, C161Y and R163W mutations reported in EE patients. The variants displayed lower iron content (1.4-to 11-fold) and lower thermal stability (1.2-to 1.7-fold) than wild-type PDO. They also exhibited varying degrees of catalytic impairment; the k cat /K m values for R163W, L55P and C161Y PDOs were 18-, 42-and 65-fold lower, respectively and the T136A variant was most affected with a 200-fold lower k cat /K m . Like wild-type enzyme, these variants were inhibited by GHcySH. This study provides the first characterization of an intermediate in the PDO-catalyzed reaction and reports on deficits associated with EE-linked mutations that are distal from the active site.Ethylmalonic encephalopathy (EE) 1 is an autosomal recessive disorder that is associated with pathological effects in the brain, gastrointestinal tract and peripheral vessels (1-3). It results in acrocyanosis, petechiae, hemorrhagic diarrhea, developmental delay and progressive neurological failure leading to necrotic lesions in the deep gray matter of the brain. Patients with EE usually succumb to the disease within the first decade of life (4). The clinical profile of EE includes elevated ethylmalonic acid in urine, C4 and C5 acrylcarnitines in blood and a deficiency of cytochrome c oxidase in brain and muscle (5). EE is caused by mutations in the ethe1 gene that encodes persulfide dioxygenase (PDO or ETHE1). Over 20 mutations have been described in the ethe1 gene, of which a subset represents missense mutations (3,4,6).PDO is a mitochondrial matrix protein that participates in the mitochondrial sulfide oxidation pathway, which converts H 2 S to the end products, thiosulfate and sulfate (7,8). In addition to PDO, the other enzymes involved in the mitochondrial sulfur oxidation pathway are sulfide quinone oxidoreductase (9), rhodanese (10), and sulfite oxidase (11). PDO catalyzes the second step in the pathway, i.e. the oxidation of glutathione persulfide (GSSH) to sulfite (Eq. 1) (12). Sulfite is either oxidized b...

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Cryptic Sulfur Incorporation in Thioangucycline Biosynthesis

et al. 2021

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Sulfur incorporation into natural products is a critical area of biosynthetic studies. Recently, a subset of sulfur‐containing angucyclines has been discovered, and yet, the sulfur incorporation step is poorly understood. In this work, a series of thioether‐bridged angucyclines were discovered, and a cryptic epoxide Michael acceptor intermediate was revealed en route to thioangucyclines (TACs) A and B. However, systematic gene deletion of the biosynthetic gene cluster (BGC) by CRISPR/Cas9 could not identify any gene responsible for the conversion of the epoxide intermediate to TACs. Instead, a series of in vitro and in vivo experiments conclusively showed that the conversion is the result of two non‐enzymatic steps, possibly mediated by endogenous hydrogen sulfide. Therefore, the TACs are proposed to derive from a detoxification process. These results are expected to contribute to the study of both angucyclines and the utilization of inorganic sulfur in natural product biosynthesis.

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Structural and biochemical analyses indicate that a bacterial persulfide dioxygenase–rhodanese fusion protein functions in sulfur assimilation

Cited by 32 publications

References 51 publications

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

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

Mechanism-based inhibition of human persulfide dioxygenase by γ-glutamyl-homocysteinyl-glycine

Cryptic Sulfur Incorporation in Thioangucycline Biosynthesis

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