hchA E.Coli
Description
Functional Roles
Hsp31 operates as a molecular chaperone and deglycase enzyme, addressing distinct stress-related challenges:
Molecular Chaperone Activity
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Holdase Function: Stabilizes partially unfolded proteins during thermal stress, preventing aggregation .
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Cooperation with DnaK-DnaJ-GrpE: Manages protein misfolding under extreme conditions (e.g., heat shock) .
Enzymatic Activities
Deglycation: Removes advanced glycation end-products (AGEs) from proteins, such as N-ω-(1-hydroxy-2-oxopropyl)-arginine and S-(1-hydroxy-2-oxopropyl)-cysteine .
Regulatory Mechanisms
Hsp31 expression is tightly controlled by stress-responsive regulators:
Key Findings:
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Heat Shock: H-NS dissociation from the promoter allows σ<sup>D</sup>-mediated transcription .
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Stationary Phase: σ<sup>S</sup> upregulates hchA transcription, enhancing survival under nutrient deprivation .
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Transcript Stability: Elevated temperatures increase hchA mRNA stability, prolonging protein production .
Physiological Impact
Hsp31 is critical for stress resilience, particularly in stationary-phase E. coli:
Mechanistic Insights:
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Acid Resistance: Hsp31 stabilizes acid-denatured proteins, supporting AR1 (periplasmic chaperones) and AR2/AR3 (decarboxylation systems) .
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MG Detoxification: Glyoxalase III activity prevents MG-induced protein cross-linking and DNA damage .
Biochemical Properties
| Parameter | Value |
|---|---|
| Optimal pH | 6.0–8.0 |
| Optimal Temperature | 35–40°C |
| Inhibitors | Cu²⁺, Fe³⁺, Zn²⁺ (blocking glyoxalase activity) |
| Cofactors | None required for glyoxalase activity |
Catalytic Mutagenesis:
Applications and Research Utility
Recombinant Hsp31 is widely used to study:
Product Specs
Chaperone protein hchA, EcHsp31, Hsp31, hchA, yedU, yzzC, b1967, JW1950.
MGSSHHHHHH SSGLVPRGSH MTVQTSKNPQ VDIAEDNAFF PSEYSLSQYT SPVSDLDGVD YPKPYRGKHK ILVIAADERY LPTDNGKLFS TGNHPIETLL PLYHLHAAGF EFEVATISGL MTKFEYWAMP HKDEKVMPFF EQHKSLFRNP KKLADVVASL NADSEYAAIF VPGGHGALIG LPESQDVAAA LQWAIKNDRF VISLCHGPAA FLALRHGDNP LNGYSICAFP DAADKQTPEI GYMPGHLTWY FGEELKKMGM NIINDDITGR VHKDRKLLTG DSPFAANALG KLAAQEMLAA YAG.
Q&A
What is the hchA gene in E. coli and what protein does it encode?
The hchA gene in Escherichia coli K-12 encodes Hsp31 (Heat shock protein 31), a 283-amino acid protein that functions as a heat-inducible molecular chaperone. The protein contains a putative catalytic triad consisting of Asp-214, His-186, and Cys-185, which is essential for its enzymatic activities . Recombinant Hsp31 can be expressed with affinity tags (such as His-tags) to facilitate purification for biochemical and structural studies .
What are the primary biochemical functions of Hsp31?
Hsp31 has been characterized with several distinct biochemical activities:
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Glyoxalase III activity: Catalyzes the direct conversion of methylglyoxal (MG) to D-lactate without requiring additional cofactors .
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Protein and nucleotide deglycase activity: Repairs methylglyoxal- and glyoxal-glycated proteins and nucleotides by deglycating cysteine, arginine, and lysine residues .
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Molecular chaperone function: Assists in protein folding under stress conditions, consistent with its classification as a heat shock protein .
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Weak aminopeptidase activity: Demonstrates limited peptidase function, though this is not its primary role .
How does Hsp31 function as glyoxalase III?
Hsp31 functions as glyoxalase III by directly converting methylglyoxal to D-lactate in a single enzymatic step without requiring cofactors. The enzyme exhibits Michaelis-Menten kinetics for methylglyoxal with a Km of 1.43±0.12 mM and a kcat of 156.9±5.5 min−1 . This mechanism differs significantly from the conventional glyoxalase system (glyoxalase I and II), which requires glutathione as a cofactor and operates through a two-step process .
| Property | Hsp31 (Glyoxalase III) | Classical Glyoxalase System (I/II) |
|---|---|---|
| Cofactor requirement | None | Glutathione |
| Enzymatic steps | Single step | Two enzymes, two steps |
| Key residues | Cys-185, His-186, Asp-214, Glu-77 | Varies by organism |
| Product | D-lactate | D-lactate |
| Intermediate | None | S-D-lactoylglutathione |
What are the optimal conditions for studying Hsp31's glyoxalase activity?
The enzymatic activity of Hsp31 is influenced by several experimental conditions:
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Inhibitory factors: Activity significantly inhibited by Cu²⁺, Fe³⁺, and Zn²⁺
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Expression conditions: Production stimulated by stationary phase, anaerobiosis, high temperature, and nutrient depletion
Researchers should consider these parameters when designing activity assays to ensure optimal enzyme performance.
How can researchers experimentally demonstrate Hsp31's deglycase activity?
To assess Hsp31's deglycase activity, researchers can employ several methodological approaches:
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Protein deglycase assay: Prepare glycated model proteins (serum albumin, aspartate aminotransferase, glyceraldehyde-3-phosphate dehydrogenase, or fructose biphosphate aldolase) by incubation with methylglyoxal. After treating with purified Hsp31, measure the restoration of enzymatic activity or reduction in glycation adducts .
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Nucleotide deglycase assay: Treat nucleotides (GTP, GDP, GMP, dGTP) with methylglyoxal or glyoxal, then incubate with Hsp31 and analyze repair using chromatographic or mass spectrometry techniques .
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Product detection: Quantify the formation of lactate or glycolate (products of methylglyoxal or glyoxal deglycation) using enzymatic assays or HPLC.
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In vivo assessment: Compare glycation levels in wild-type versus hchA-knockout strains using immunological methods specific for glycation adducts.
What experimental approaches can be used to study hchA function in E. coli?
Several sophisticated experimental approaches can be employed to investigate hchA function:
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Gene deletion studies: Creating hchA-deficient strains reveals that cells become more susceptible to methylglyoxal, particularly during stationary phase .
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Complementation experiments: Reintroducing plasmid-encoded hchA into knockout strains reverses susceptibility phenotypes, confirming the specific protective role of this gene .
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Site-directed mutagenesis: Targeted mutations in catalytic residues reveal that Cys-185 and Glu-77 are essential for catalysis, while His-186 contributes but is not absolutely required .
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Recombinant protein expression: Expression with affinity tags facilitates purification for biochemical and structural studies .
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Structural biology approaches: X-ray crystallography has revealed the catalytic triad and potential binding modes for substrates.
How do mutations in the catalytic residues affect Hsp31 function?
Mutagenesis studies have provided critical insights into the catalytic mechanism of Hsp31:
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Cys-185: Mutation completely abolishes glyoxalase activity, indicating this residue is essential and likely serves as the primary nucleophile in the catalytic mechanism .
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His-186: Mutation reduces but does not eliminate enzymatic activity, suggesting it participates in catalysis but is not absolutely essential, unlike in many classical catalytic triads .
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Glu-77: Though not part of the traditional catalytic triad, this residue is essential for catalysis, with mutations abolishing enzymatic activity .
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Asp-214: Part of the putative catalytic triad, likely stabilizes the protonated form of His-186 during catalysis.
These findings suggest Hsp31 utilizes a somewhat atypical catalytic mechanism compared to classical cysteine-dependent enzymes.
What are the effects of hchA deletion on E. coli physiology?
Studies of hchA-deficient E. coli strains have revealed several important physiological consequences:
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Increased methylglyoxal sensitivity: Stationary-phase E. coli cells become more vulnerable to methylglyoxal toxicity when hchA is deleted .
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Intracellular methylglyoxal accumulation: Knockout strains show higher levels of intracellular methylglyoxal compared to wild-type strains .
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Impaired stress response: Given Hsp31's heat-inducibility, its absence affects cellular resilience to various stressors.
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Glycation damage accumulation: Without Hsp31's deglycase activity, cells accumulate glycated proteins and nucleotides, potentially leading to dysfunction .
Complementation with plasmid-encoded hchA reverses these phenotypes, confirming the specific role of this gene in cellular protection .
What role does Hsp31 play in E. coli stress response pathways?
Hsp31 contributes to stress response through multiple mechanisms:
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Methylglyoxal detoxification: As glyoxalase III, it detoxifies methylglyoxal which accumulates under various stress conditions including high glucose, stationary phase, and oxidative stress .
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Prevention of glycation damage: By repairing glycated proteins and nucleotides, it prevents formation of advanced glycation end products (AGEs) that cause irreversible cellular damage .
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Heat stress adaptation: As a heat-inducible protein, it likely protects cellular components during thermal stress through its chaperone activity.
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Stationary phase survival: Particularly important for methylglyoxal resistance during stationary phase, suggesting a role in long-term survival and stress adaptation .
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Guanine glycation repair: Participates in nucleotide repair systems that reverse methylglyoxal and glyoxal damage via nucleotide sanitization .
How does Hsp31's glyoxalase activity compare with other glyoxalase systems?
The glyoxalase activity of Hsp31 (glyoxalase III) differs fundamentally from the canonical glyoxalase system:
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Reaction mechanism: Hsp31 performs direct conversion of methylglyoxal to D-lactate in a single step, whereas the canonical system uses two enzymes (glyoxalase I and II) and produces an intermediate .
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Cofactor requirements: Hsp31 functions independently without cofactors, while the canonical system requires glutathione .
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Evolutionary implications: The presence of multiple systems for methylglyoxal detoxification suggests evolutionary redundancy for this critical cellular function.
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Stress adaptation: The cofactor-independent nature of Hsp31 may provide an advantage under conditions where glutathione is depleted.
How can researchers distinguish between the contributions of different glyoxalase systems in E. coli?
To differentiate between the activities of different glyoxalase systems in E. coli, researchers can employ several methodological approaches:
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Genetic manipulation: Create single and combined knockouts of genes encoding different glyoxalases (glyoxalase I, glyoxalase II, and hchA) to assess relative contributions to methylglyoxal resistance.
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Enzyme inhibition studies: Use specific inhibitors for different glyoxalase systems and measure remaining methylglyoxal detoxification activity.
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Cofactor depletion: Deplete glutathione in cells to inhibit the canonical pathway while leaving Hsp31 function intact.
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Expression analysis: Monitor expression patterns of different glyoxalases under various stress conditions to identify specialized roles.
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Biochemical assays: Measure the activity of each system in cell-free extracts using specific substrates and conditions that favor one system over others.
Product Science Overview
Chaperone proteins play a crucial role in the cellular machinery by assisting the folding of newly synthesized proteins, preventing aggregation, and refolding misfolded proteins. One such chaperone protein is hchA, found in Escherichia coli (E. coli). The recombinant form of this protein has been extensively studied for its potential applications in biotechnology and medicine.
The hchA protein, also known as heat shock protein Hsp31, belongs to the DJ-1/ThiJ/PfpI superfamily. It is a small molecular chaperone that is upregulated in response to heat shock and other stress conditions. The primary function of hchA is to prevent the aggregation of proteins under stress conditions, thereby maintaining cellular homeostasis.
The recombinant production of hchA involves the insertion of the hchA gene into an expression vector, which is then introduced into E. coli cells. The cells are cultured under conditions that induce the expression of the recombinant protein. The protein is then purified using techniques such as affinity chromatography, which exploits the specific binding properties of the protein to isolate it from other cellular components.
The activity of hchA can be analyzed through various biochemical assays that measure its ability to prevent protein aggregation. One common method is the use of a model substrate protein that tends to aggregate under stress conditions. The presence of hchA can significantly reduce the aggregation of this substrate, demonstrating its chaperone activity.
Additionally, structural analysis techniques such as X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy can be employed to determine the three-dimensional structure of hchA. These studies provide insights into the molecular mechanisms by which hchA interacts with substrate proteins and prevents their aggregation.
The recombinant hchA protein has potential applications in various fields. In biotechnology, it can be used to improve the yield and solubility of other recombinant proteins by co-expressing it with target proteins. In medicine, hchA and other chaperone proteins are being explored for their potential to treat diseases associated with protein misfolding and aggregation, such as neurodegenerative disorders.
