Recombinant 3-mercaptopyruvate sulfurtransferase (sseA)
Description
Enzymatic Function and Catalytic Mechanism
Recombinant MPST catalyzes the transfer of sulfur atoms from 3-mercaptopyruvate (3MP) to acceptor molecules, such as cyanide or thiol compounds, via a ping-pong mechanism . The reaction proceeds in two steps:
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Desulfuration: 3MP is converted into pyruvate, leaving a persulfidated active-site cysteine (Cys247) .
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Transpersulfidation: The outer sulfur atom is transferred to nucleophilic acceptors (e.g., glutathione or cyanide) .
This enzyme contributes to:
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Hydrogen sulfide biosynthesis: MPST works alongside cysteine aminotransferase (CAT) to generate H₂S from cysteine-derived 3MP .
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Cyanide detoxification: Converts toxic cyanide into less harmful thiocyanate .
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Protein persulfidation: Regulates redox signaling by modifying cysteine residues in target proteins .
Recombinant Production and Applications
Recombinant MPST is typically expressed in E. coli systems for high-yield purification . Key applications include:
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Drug discovery: Used to screen inhibitors targeting H₂S signaling pathways .
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Disease modeling: Studying MPST's role in metabolic disorders, cancer, and neurodegenerative diseases .
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Biochemical assays: Quantifying enzyme kinetics and persulfidation dynamics .
Inhibitors and Selectivity
Recent studies have identified selective inhibitors for recombinant MPST:
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Compound 3: A pyrimidone derivative with an IC₅₀ of 0.18 µM for mouse MPST; >100-fold selectivity over rhodanese .
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HMPSNE: Inhibits human MPST with IC₅₀ ~30 µM; reduces H₂S production in cancer cells .
Table 3: Inhibitor Profiles
| Inhibitor | IC₅₀ (MPST) | Selectivity vs. Rhodanese | Cellular Activity |
|---|---|---|---|
| Compound 3 | 0.18 µM | >100-fold | Yes (COS7 cells) |
| HMPSNE | 30 µM | Not reported | Yes (CT26 cells) |
Research Findings
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Cancer: MPST inhibition suppresses proliferation and migration in colon cancer cells (CT26), likely via disruption of mitochondrial bioenergetics .
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Oxidative stress: MPST knockout models show reduced resistance to oxidative damage, linking the enzyme to redox homeostasis .
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Neuroprotection: MPST-derived H₂S modulates neurotransmission and mitigates neuronal apoptosis .
Clinical and Therapeutic Implications
Product Specs
Target Background
KEGG: ece:Z3788
STRING: 155864.Z3788
Q&A
What is 3-mercaptopyruvate sulfurtransferase (sseA) and what distinguishes it from other sulfurtransferases?
3-Mercaptopyruvate sulfurtransferase (sseA) is a 31 kDa protein that catalyzes the transfer of sulfur from 3-mercaptopyruvate to various acceptor molecules, including cyanide. It represents the prototype of a distinct sulfurtransferase subfamily that differs from the better-characterized rhodanese sulfurtransferases, which primarily display thiosulfate:cyanide sulfurtransferase activity . Both enzyme families share a catalytic mechanism centered on a reactive invariant cysteine residue, but their substrate preferences and reaction mechanisms differ significantly . In E. coli, sseA is encoded by the sseA gene and has been crystallized in tetragonal form (space group P4(1) or P4(3)) with unit-cell parameters a = b = 150.2, c = 37.9 Å .
How does 3MST contribute to hydrogen sulfide and sulfane sulfur production?
3MST produces hydrogen sulfide (H₂S) from 3-mercaptopyruvate (3MP), which is generated from L-cysteine and α-ketoglutarate by cysteine aminotransferase (CAT) . Recent studies have revealed that 3MST also generates sulfane sulfur species (H₂Sₙ), which are sulfur atoms with oxidation states 0 or -1 . These sulfane sulfurs, rather than H₂S itself, may be the actual mediators of certain physiological processes previously attributed to H₂S . The production of H₂S by 3MST requires reducing equivalents, with thioredoxin and dihydrolipoic acid serving as essential cofactors for this activity .
What is the catalytic mechanism of 3MST?
The 3MST-catalyzed reaction with 3-mercaptopyruvate proceeds via a ping-pong mechanism . Initially, the enzyme's catalytic cysteine residue attacks the substrate 3-mercaptopyruvate, forming a persulfurated enzyme intermediate. This persulfurated cysteine then transfers the sulfur atom to an acceptor molecule in the second half of the reaction . X-ray crystallography studies of 3MST-inhibitor complexes have confirmed that inhibitors target this persulfurated cysteine residue in the active site . Computational analyses have demonstrated a strong long-range electrostatic interaction between the persulfur anion of the persulfurated cysteine and the positively charged carbonyl carbon of inhibitor molecules .
What expression systems are most effective for producing recombinant 3MST (sseA)?
Recombinant 3-mercaptopyruvate sulfurtransferase (sseA) can be expressed and purified from multiple host systems, each with distinct advantages:
| Host System | Advantages | Disadvantages | Applications |
|---|---|---|---|
| E. coli | High yield, rapid production, cost-effective | Limited post-translational modifications | Structural studies, initial activity screening |
| Yeast | Good yield, some post-translational modifications | Longer production time than E. coli | Functional studies requiring some modifications |
| Insect cells | Better post-translational modifications | Lower yield, more complex | Studies requiring properly folded protein |
| Mammalian cells | Most complete post-translational modifications | Lowest yield, most expensive, longest timeline | Studies requiring native-like activity |
For most research applications, E. coli and yeast expression systems offer the best balance of yield and turnaround time .
What strategies optimize the functional activity of recombinant 3MST?
Optimizing functional activity of recombinant 3MST requires careful consideration of the reactive cysteine residue in the active site. The persulfurated catalytic cysteine is essential for 3MST activity and is highly susceptible to oxidation . Successful expression strategies should:
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Include reducing agents in all purification buffers
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Minimize exposure to oxidizing conditions
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Consider fusion tags that improve solubility without affecting the active site
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Verify catalytic activity using standard assays with 3-mercaptopyruvate as substrate
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For bacterial 3MST (such as E. coli SseA), optimize expression conditions to ensure proper folding of the 31 kDa protein
The reactive invariant cysteine must maintain its nucleophilicity for the enzyme to function properly in experimental conditions .
What are the established methods for measuring 3MST activity?
Several complementary approaches can be used to measure 3MST activity:
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Fluorescent probe-based assays: Hydrogen sulfide-selective fluorescent probes such as HSip-1 have been successfully used in high-throughput screening to identify 3MST inhibitors . These probes enable real-time monitoring of H₂S production.
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Cyanide assay: Measuring 3-mercaptopyruvate:cyanide sulfurtransferase activity by quantifying the conversion of cyanide to thiocyanate .
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LC-MS/MS detection: High-performance liquid chromatography tandem mass spectrometry can be used to directly measure 3-mercaptopyruvate concentrations in biological samples .
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Polysulfide detection assays: Specific assays have been developed to quantify polysulfide (H₂Sₙ) production by 3MST in biological fluid samples .
How can researchers distinguish between H₂S and sulfane sulfur production by 3MST?
Distinguishing between H₂S and sulfane sulfur (H₂Sₙ) production requires specific analytical approaches:
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Use sulfane sulfur-specific fluorescent probes such as SSP series compounds that react selectively with sulfane sulfur species .
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Employ combined assay approaches that can quantitatively determine polysulfides in biological samples, including those developed for albumins and plasma proteins .
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Analyze reaction products using mass spectrometry to identify H₂S₃ and other polysulfide species produced by 3MST .
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Compare results with and without reducing agents, as some sulfane sulfur species are stable only under specific redox conditions .
Research has shown that E. coli utilizes separate enzymes to produce H₂S and reactive sulfane sulfur from L-cysteine, with 3MST playing a distinct role in this process .
What structural features characterize effective 3MST inhibitors?
High-throughput screening of large chemical libraries (174,118 compounds) using H₂S-selective fluorescent probes has identified several potent 3MST inhibitors . Effective inhibitors share these common structural features:
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Aromatic ring-carbonyl-S-pyrimidone structure
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Positively charged carbonyl carbon in the pyrimidone moiety
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Ability to interact with the persulfurated cysteine residue in the active site
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Proper molecular geometry to fit the enzyme's active site
Compound 3, identified in these studies, demonstrated very high selectivity for 3MST over other H₂S/sulfane sulfur-producing enzymes and rhodanese . X-ray crystallography confirmed that these inhibitors specifically target the persulfurated cysteine residue located in the active site of 3MST .
How do inhibitor-enzyme interactions at the molecular level inform drug design?
X-ray crystal structures of 3MST complexes with inhibitors have revealed crucial insights into inhibitor binding mechanisms:
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Inhibitors target the persulfurated cysteine residue in the enzyme's active site .
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A strong long-range electrostatic interaction exists between the persulfur anion of the persulfurated cysteine residue and the positively charged carbonyl carbon of the pyrimidone moiety in effective inhibitors .
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This interaction provides a structural basis for designing more potent and selective 3MST inhibitors.
These molecular details have informed the development of bacterial 3MST inhibitors that synergistically control bacterial survival when combined with other antimicrobials .
What role does 3MST play in bacterial defense mechanisms?
3MST contributes significantly to bacterial defense mechanisms through hydrogen sulfide production:
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H₂S serves as a bacterial defense mechanism against host immune responses .
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H₂S production provides a universal defense against antibiotics in bacteria .
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In E. coli, H₂S mediates protection against oxidative stress, which is a key component of many antibiotic mechanisms of action .
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The transcription factor YcjW controls emergency H₂S production in E. coli .
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H₂S contributes to redox balancing against genetic perturbations and modulates central carbon metabolism under oxidative stress conditions .
These findings suggest that targeting 3MST could potentially enhance antibiotic efficacy by disrupting bacterial defense mechanisms .
How does bacterial 3MST function differ from human 3MST?
Understanding the differences between bacterial and human 3MST is crucial for developing selective antimicrobial strategies:
These differences provide opportunities for developing targeted antimicrobial approaches that inhibit bacterial 3MST without affecting human enzyme function .
How can 3MST be utilized in developing novel antimicrobial strategies?
3MST represents a promising target for novel antimicrobial strategies based on several key findings:
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Inhibition of bacterial 3MST can synergistically enhance the efficacy of existing antibiotics by disrupting H₂S-mediated defense mechanisms .
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H₂S production by 3MST contributes to bacterial resistance against oxidative stress imposed by antibiotics, making it a valuable adjuvant target .
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Selective inhibitors can be designed that target bacterial 3MST while minimizing effects on human homologs, potentially reducing side effects .
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Combining 3MST inhibitors with antibiotics that induce oxidative stress could create synergistic antimicrobial effects by simultaneously increasing oxidative damage while blocking protective H₂S production .
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The "on demand" redox buffering provided by H₂S contributes to antibiotic resistance, suggesting that 3MST inhibition could restore sensitivity to existing antibiotics .
What experimental approaches can elucidate the persulfidation mechanism of 3MST?
To investigate the persulfidation mechanism of 3MST, researchers can employ these advanced approaches:
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Site-directed mutagenesis: Systematically modify the catalytic cysteine and surrounding residues to understand their contributions to persulfide formation and transfer .
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Time-resolved crystallography: Capture intermediate states of the enzyme during the catalytic cycle to visualize persulfide formation and transfer .
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Quantum mechanics/molecular mechanics (QM/MM) simulations: Perform precise theoretical calculations to elucidate the electronic structure and energetics of persulfide formation and electrostatic interactions with inhibitors .
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Mass spectrometry of intact enzyme and peptides: Identify and quantify persulfidated cysteine residues under different conditions .
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Alternative pathway analysis: Investigate the pathway of H₂S and polysulfides production from sulfurated catalytic-cysteine of reaction intermediates .
Recent studies have begun unraveling the mechanism of cysteine persulfide formation catalyzed by 3-mercaptopyruvate sulfurtransferases, providing a foundation for these investigations .
