Recombinant Chicken Alcohol dehydrogenase [NADP (+)] (AKR1A1)

What is AKR1A1 and what are its primary biological functions?

AKR1A1 is the founding member of the aldo-keto reductase superfamily, with orthologs found throughout the vertebrate subphylum. It primarily catalyzes the reduction of aldehyde groups to their corresponding alcohols in an NADPH-dependent manner . This enzyme serves multiple critical biological functions:

• Acts as an S-nitroso-glutathione reductase (GSNOR) and SNO-CoA reductase, regulating protein S-nitrosylation patterns throughout cells

• Provides protection against alcohol-associated liver disease (ALD) by reducing the accumulation of toxic aldehydes like 4-hydroxynonenal (4-HNE)

• Inhibits 4-HNE-mediated p53 activation, which contributes to its protective role against liver injury

• Participates in drug metabolism pathways for compounds such as doxorubicin and tiaprofenic acid

• Contributes to prostaglandin F2 biosynthesis

• Catalyzes the conversion of glucuronate (GlucA) to gulonate, a key step in L-ascorbic acid synthesis in some species

In mammals, AKR1A1 expression appears tissue-dependent, with significant presence in kidneys, liver, lungs, and spleen, corresponding to its protective functions in these organs .

How is AKR1A1 enzymatic activity measured in laboratory settings?

AKR1A1 activity can be measured through several established methodologies, with the NADPH consumption assay being the most widely used approach:

NADPH Spectrophotometric Assay:
The standard reaction mixture contains 100 mM HEPES buffer (pH 7.4), 0.1 mM NADPH, and an appropriate substrate concentration (e.g., 10 mM glucuronate or other substrate) . The decrease in absorbance at 340 nm is monitored over time as NADPH is oxidized to NADP+. Enzyme activity is typically defined as the amount that catalyzes the oxidation of 1 μmol of NADPH per minute .

Activity Calculation:
Enzyme activity = (ΔA340/min) × (total volume) / (ε × path length × enzyme volume)
Where ε is the extinction coefficient of NADPH (6,220 M⁻¹cm⁻¹)

For S-nitrosothiol reduction assays, additional methods include:

• Griess assay for measuring nitrite formation

• Chemiluminescence detection for NO released during reduction

• SNO-RAC techniques to assess protein S-nitrosylation levels before and after enzyme treatment

When working with tissue samples, immunodepletion of AKR1A1 can be used as a control to verify the specificity of measured activity. This approach has demonstrated that AKR1A1 accounts for the majority of NADPH-dependent GSNO reductase activity in kidney tissues .

What substrates does AKR1A1 act upon and what is its substrate preference profile?

AKR1A1 demonstrates activity toward a diverse range of substrates, with distinct preference patterns:

Primary Substrates:

• S-nitrosothiols:

  • S-nitroso-glutathione (GSNO)

  • S-nitroso-CoA (SNO-CoA)

  • Kinetic studies suggest a substrate preference of SNO-CoA > GSNO

• Sugar derivatives:

  • D-glucuronate (GlucA) - particularly relevant in L-ascorbic acid biosynthesis

• Lipid peroxidation products:

  • 4-hydroxynonenal (4-HNE), a toxic aldehyde produced during oxidative stress

• Other aldehydes and ketones:

  • Various physiological and xenobiotic compounds containing carbonyl groups

Substrate preference can be influenced by mutations in key residues. For example, molecular modeling and mutagenesis studies have identified Arg-312 as critical for GSNO reduction, while Lys-127 primarily affects SNO-CoA reduction . These distinct binding determinants explain how AKR1A1 can differentially regulate these two major low-molecular-weight S-nitrosothiols.

What experimental conditions optimize recombinant AKR1A1 activity?

Optimizing conditions for recombinant chicken AKR1A1 activity requires careful attention to several parameters:

Buffer and pH:

• HEPES buffer (100 mM, pH 7.4) is commonly used for AKR1A1 assays

• Optimal pH range is typically 7.0-7.5 for most substrates

• Phosphate buffers may also be suitable but may interfere with some metal-dependent processes

Temperature:

• Standard assays are conducted at 37°C to mimic physiological conditions

• Chicken AKR1A1 may have slightly different temperature optima reflecting the higher body temperature of birds (40-42°C)

Cofactor requirements:

• NADPH is the essential cofactor, typically used at 0.1-0.5 mM concentrations

• Fresh preparation of NADPH is recommended as it degrades over time

Substrate concentrations:

• For kinetic studies, substrate ranges should span approximately 0.1-10× Km values

• For GSNO, concentrations of 0.1-2 mM are typically appropriate

• For GlucA, higher concentrations (5-20 mM) may be needed due to higher Km values

Assay duration and enzyme concentration:

• Initial velocity measurements should be in the linear range (typically first 10-20% of reaction)

• Enzyme amount should be adjusted to achieve measurable rates within this linear range

• For recombinant preparations, protein purity should exceed 95% to minimize interfering activities

When working with S-nitrosothiol substrates, special precautions include protection from light and preparation of fresh solutions immediately before use to prevent spontaneous decomposition.

How does expression system choice affect recombinant AKR1A1 properties?

The choice of expression system significantly impacts the properties and performance of recombinant chicken AKR1A1:

E. coli expression systems:

• Advantages: High yield, simplicity, cost-effectiveness

• Limitations: Lack of post-translational modifications, potential inclusion body formation

• Optimization: Codon optimization for E. coli, expression at lower temperatures (16-25°C), use of solubility tags (MBP, SUMO)

• Common vectors: pET series, pGEX for GST fusion

Insect cell expression (Baculovirus):

• Advantages: Better folding, some post-translational modifications, higher solubility

• Limitations: More complex, longer production time, higher cost

• Best for: Obtaining enzymes with native-like activity when E. coli systems fail

• Systems: Sf9, High Five cells with vectors like pFastBac

Mammalian expression:

• Advantages: Most authentic post-translational modifications, proper folding

• Limitations: Lower yield, highest cost, most complex

• Best for: Studies focusing on regulatory modifications of AKR1A1

• Systems: HEK293, CHO cells with vectors like pcDNA

Yeast expression:

• Advantages: Eukaryotic processing, high yield, relatively simple

• Limitations: Hyper-glycosylation can occur, different codon bias

• Systems: Pichia pastoris, Saccharomyces cerevisiae

Key considerations for purification include:

• Affinity tags: His6, GST, or FLAG tags facilitate purification but may affect activity

• Tag removal: Inclusion of protease cleavage sites allows tag removal after purification

• Buffer optimization: Including glycerol (10-20%) and reducing agents improves stability

• Storage: Small aliquots at -80°C prevent repeated freeze-thaw cycles

How does AKR1A1 contribute to protection against alcohol-associated liver disease?

AKR1A1 demonstrates significant protective effects against alcohol-associated liver disease (ALD) through several key mechanisms:

Reduction of toxic aldehydes:
Knockout studies in mice have shown that AKR1A1 protects against alcohol-induced liver damage by reducing the accumulation of 4-hydroxynonenal (4-HNE), a toxic aldehyde produced during lipid peroxidation . Alcohol-fed Akr1a1-/- mice displayed more severe liver injury than wild-type counterparts, with increased proinflammatory cytokines, oxidative stress markers, and lipid accumulation in their livers .

Inhibition of 4-HNE-mediated p53 activation:
AKR1A1 prevents 4-HNE-induced activation of p53, a key mediator of cell death pathways. Loss of AKR1A1 leads to increased 4-HNE accumulation and subsequent p53 phosphorylation, contributing to liver injury progression . This suggests that AKR1A1's protective role involves not just detoxification but also modulation of critical cellular signaling pathways.

Regulation of inflammatory responses:
Experimental evidence shows that Akr1a1-/- mice exhibit increased proinflammatory cytokine production in response to alcohol feeding . This suggests AKR1A1 may modulate inflammatory processes either directly or through its effects on oxidative stress.

Protection against fibrosis:
Alcohol-fed Akr1a1-/- mice develop more severe liver fibrosis compared to wild-type controls . In vitro studies using AML12 hepatocytes found that Akr1a1 knockdown aggravated fibrosis induced by TGF-β1, confirming the enzyme's protective role against fibrotic processes .

Antioxidant defense system support:
AKR1A1 appears to support the broader antioxidant defense system, as Akr1a1-/- mice show reduced levels of antioxidant enzymes in their livers following alcohol exposure . This suggests AKR1A1 may indirectly influence the expression or activity of other protective enzymes.

The clinical relevance of these findings is underscored by observations that AKR1A1 is downregulated in patients diagnosed with ALD , suggesting that loss of this protective mechanism may contribute to disease progression in humans.

What structural features determine AKR1A1's substrate specificity for S-nitrosothiols?

AKR1A1's ability to reduce both S-nitroso-glutathione (GSNO) and S-nitroso-CoA (SNO-CoA) is determined by specific structural features that enable substrate recognition and catalysis:

Key binding residues for GSNO reduction:
Molecular modeling and mutagenesis studies have identified Arg-312 as a critical residue specifically mediating the interaction with GSNO . This positively charged residue likely forms ionic interactions with the carboxyl groups of glutathione. When Arg-312 is mutated, GSNO reductase activity is dramatically reduced while other catalytic functions remain intact.

SNO-CoA binding determinants:
Lys-127 has been identified as important for SNO-CoA binding, with substitution of this residue having minimal effect on GSNO reduction but significantly impairing SNO-CoA reductase activity . This suggests that AKR1A1 utilizes distinct binding regions for different S-nitrosothiol substrates.

Substrate preference mechanism:
Kinetic analyses indicate that AKR1A1 has a substrate preference of SNO-CoA > GSNO . This preference appears to be determined by:

• The specific arrangement of charged residues in the binding pocket

• Complementarity between substrate structure and binding site topography

• Differential hydrogen bonding networks formed with each substrate

Catalytic core architecture:
The active site contains the conserved catalytic tetrad typical of aldo-keto reductases that is responsible for hydride transfer from NADPH to the substrate. This core catalytic machinery accommodates diverse substrates while maintaining efficient reduction capacity.

NADPH binding domain:
The positioning of the NADPH cofactor relative to the substrate binding site is crucial for efficient catalysis. Conserved residues form specific interactions with NADPH, orienting it appropriately for hydride transfer.

These structural features enable AKR1A1 to function as a multi-LMW-SNO reductase that can distinguish between and metabolize both major low-molecular-weight S-nitrosothiol signaling molecules, allowing for nuanced control of protein S-nitrosylation under both physiological and pathological conditions .

How does AKR1A1 function compare between avian and mammalian species?

Comparison of avian (chicken) and mammalian AKR1A1 reveals important similarities and differences that reflect evolutionary adaptations:

Conserved catalytic mechanism:
The core catalytic mechanism involving NADPH-dependent reduction of carbonyl groups appears highly conserved between avian and mammalian AKR1A1 enzymes. Both utilize the same catalytic tetrad arrangement typical of the aldo-keto reductase superfamily.

S-nitrosothiol reductase activity:
Both chicken and mammalian AKR1A1 function as S-nitrosothiol reductases, able to reduce GSNO and SNO-CoA in an NADPH-dependent manner . This conservation suggests the fundamental importance of this activity across vertebrate evolution.

Substrate preference patterns:
While both can reduce similar substrates, subtle differences in substrate preference may exist due to species-specific variations in the substrate binding pocket. This could reflect different physiological demands between birds and mammals.

Tissue distribution differences:
In mammals, AKR1A1 shows high expression in kidney, liver, lungs, and spleen . Avian AKR1A1 may have a different tissue distribution pattern reflecting the unique physiological demands of birds, particularly in metabolically active tissues.

Physiological roles:
Mammalian AKR1A1 plays key roles in:

• Protection against alcohol-induced liver injury

• Regulation of protein S-nitrosylation

• Protection against acute kidney injury

• Vitamin C synthesis pathway (in mice but not humans)

The avian enzyme likely serves similar protective functions but may have additional roles adapted to avian physiology, such as handling the higher oxidative stress resulting from birds' higher body temperature and metabolic rate.

Regulatory cross-talk:
In mammals, AKR1A1 expression is upregulated in tissues lacking GSNOR (another denitrosylase) , suggesting compensation between different denitrosylation systems. Similar regulatory networks may exist in avian species, though the specific patterns may differ.

Evolutionary significance:
The presence of AKR1A1 across both avian and mammalian lineages, which diverged approximately 310-330 million years ago, highlights the enzyme's fundamental importance in cellular metabolism and redox regulation throughout vertebrate evolution.

What regulatory mechanisms control AKR1A1 expression and activity?

AKR1A1 is subject to multiple levels of regulation that fine-tune its expression and activity in response to cellular conditions:

Transcriptional regulation:

• Expression levels vary significantly between tissues, with higher levels in metabolically active organs such as kidney, liver, and lungs

• Evidence from mammalian studies suggests upregulation in response to oxidative stress conditions

• Compensatory upregulation occurs in tissues lacking other denitrosylases like GSNOR, as demonstrated by increased AKR1A1 protein levels in liver extracts from GSNOR-/- mice

Post-translational modifications:
Several modifications can affect AKR1A1 activity:

• Phosphorylation of serine/threonine residues may alter catalytic activity

• S-nitrosylation of cysteine residues potentially creates a feedback mechanism where the enzyme's own substrates regulate its activity

• Oxidative modifications of key residues can reduce enzyme function during oxidative stress

Metabolic regulation:

• NADPH/NADP+ ratio influences activity, with higher NADPH availability promoting enzyme function

• Substrate availability, particularly during stressed conditions, may drive increased activity

• Competitive inhibition by similar substrates can regulate which reactions are prioritized

Genetic variation:
Genetic variants have been identified that impact function:

• The c.753G > A variant causes exon skipping, leading to a loss of gene expression and enzymatic activity

• In this variant, exon 8 skipping causes a frameshift mutation resulting in a truncated protein that completely loses enzymatic activity

• Such variants have been associated with conditions like schizophrenia, suggesting clinical relevance

Allosteric regulation:

• Binding of effector molecules at sites distinct from the active site may alter catalytic efficiency

• Conformational changes induced by protein-protein interactions could modulate activity

Protein stability and turnover:

• Regulation of protein half-life through ubiquitination and proteasomal degradation

• Chaperone interactions that affect proper folding and stability

The interplay between these regulatory mechanisms allows for precise control of AKR1A1 function across different physiological contexts. Under pathological conditions like alcohol-associated liver disease, AKR1A1 is downregulated , suggesting that dysregulation of these control mechanisms may contribute to disease progression.

How does the c.753G>A variant affect AKR1A1 function and its association with schizophrenia?

The c.753G>A variant of AKR1A1 has significant functional consequences and potential clinical implications:

Molecular consequences:
The c.753G>A variant (rs745484618) causes exon skipping during mRNA processing. Although this is a synonymous variant (p.Arg251Arg) that doesn't change the encoded amino acid, it critically affects splicing . Exon 8 skipping results in a frameshift mutation that produces a truncated protein. The normal AKR1A1 protein consists of 325 amino acids, but the variant creates a protein where only the first 88 amino acids match the normal sequence, followed by 10 altered amino acids (total of 261 amino acids) .

Functional impact:
Experimental studies with recombinant proteins demonstrate that the truncated AKR1A1 produced by the c.753G>A variant completely loses its enzymatic activity . This loss of function prevents the enzyme from performing its normal catalytic roles:

• Reduction of glucuronate (GlucA) to gulonate

• Metabolism of aldehydes and ketones

• Reduction of S-nitrosothiols

Biochemical consequences:
The variant causes GlucA accumulation in patients, as the enzyme can no longer catalyze its conversion to gulonate . This disruption may have downstream effects on multiple metabolic pathways, potentially including:

• Altered ascorbic acid metabolism (in species with functional vitamin C synthesis)

• Disrupted detoxification of reactive aldehydes

• Impaired regulation of protein S-nitrosylation

Association with schizophrenia:
The c.753G>A variant has been found in patients with schizophrenia, suggesting a potential connection between AKR1A1 function and this psychiatric disorder . Mechanistically, this association might be explained by several pathways:

• Impact on neurotransmitter metabolism:
  AKR1A1 is involved in the metabolism of noradrenaline, levels of which are reduced in patients with schizophrenia . Studies have shown that negative symptoms of schizophrenia can be improved by inhibiting noradrenaline reuptake .

• Methylglyoxal detoxification:
  AKR1A1 metabolizes methylglyoxal, a reactive carbonyl compound . Accumulation of methylglyoxal could contribute to oxidative stress and cellular damage in neural tissues.

• Protein S-nitrosylation dysregulation:
  As an S-nitrosothiol reductase, loss of AKR1A1 function could disrupt normal patterns of protein S-nitrosylation, potentially affecting signaling pathways relevant to neural function.

These findings suggest that while AKR1A1 variants may not be the primary cause of schizophrenia, they could contribute to disease pathophysiology in certain individuals by disrupting metabolic processes important for normal neurological function.

What methodological approaches are used to study AKR1A1's role in protein S-nitrosylation?

Investigating AKR1A1's function as a regulator of protein S-nitrosylation requires specialized methodological approaches:

Detection of S-nitrosylated proteins:

• SNO-RAC (S-nitrosothiol Resin-Assisted Capture): This technique allows for the isolation and identification of S-nitrosylated proteins from complex mixtures. In studies with AKR1A1, SNO-RAC with Coomassie Blue staining demonstrated that GSNO increases SNO-protein levels in tissue extracts, but this effect is reversed by NADPH addition only in AKR1A1+/+ extracts, not in AKR1A1-/- extracts .

• Biotin switch technique: This classical approach converts S-nitrosothiols to biotinylated thiols, allowing detection via streptavidin-based methods. It has been used to demonstrate AKR1A1's ability to reduce protein S-nitrosothiols.

• Mass spectrometry-based approaches: These provide site-specific identification of S-nitrosylated residues and can quantify changes in S-nitrosylation levels following AKR1A1 activity.

Enzymatic activity assays:

• NADPH consumption assay: Monitoring the decrease in absorbance at 340 nm as NADPH is oxidized to NADP+ during the reduction of S-nitrosothiols .

• Griess assay: Measures nitrite formation following S-nitrosothiol reduction, providing an indirect measure of denitrosylase activity.

• Chemiluminescence detection: Measures NO released during S-nitrosothiol reduction.

Genetic manipulation approaches:

• AKR1A1 knockout models: AKR1A1-/- mice show greatly reduced NADPH-dependent GSNO reductase activity (~90% reduction in kidney extracts), confirming AKR1A1's major role in this process .

• Immunodepletion: Removal of AKR1A1 from tissue extracts using specific antibodies significantly reduces NADPH-dependent GSNO reductase activity, providing evidence for its contribution to total cellular denitrosylase activity .

• Site-directed mutagenesis: Creation of mutants (e.g., in Arg-312 or Lys-127) to investigate residues important for S-nitrosothiol binding and reduction .

Comparative studies:

• Tissue distribution analysis: Examination of AKR1A1 expression and activity across different tissues reveals correlation with NADPH-dependent GSNO reductase activity levels .

• Cross-talk with other denitrosylases: Studies in GSNOR-/- mice show upregulation of AKR1A1, suggesting compensatory mechanisms between different denitrosylation systems .

Visualization techniques:

• Fluorescent probes for S-nitrosothiols: Allow real-time monitoring of S-nitrosothiol levels in cellular systems.

• Immunohistochemistry: Permits localization of AKR1A1 in tissue sections to correlate with areas of S-nitrosylation activity.

The combination of these approaches has established AKR1A1 as a primary mediator of NADPH-coupled GSNO reductase activity in mammalian tissues and highlighted its importance in regulating protein S-nitrosylation, a ubiquitous post-translational modification that mediates redox-based cellular signaling .

How does AKR1A1 activity relate to oxidative and nitrosative stress responses?

AKR1A1 plays multifaceted roles in cellular responses to both oxidative and nitrosative stress:

S-nitrosothiol metabolism and nitrosative stress:
AKR1A1 functions as a primary NADPH-dependent reductase for both GSNO and SNO-CoA, the major low-molecular-weight S-nitrosothiols in cells . Through this activity, AKR1A1:

• Regulates the steady-state levels of protein S-nitrosylation

• Modulates nitric oxide (NO) bioavailability

• Protects against nitrosative stress by preventing excessive S-nitrosylation of critical proteins

• Complements other denitrosylation systems like GSNOR (which is NADH-dependent)

Notably, tissues from AKR1A1-deficient mice show impaired ability to reduce SNO-protein levels when treated with GSNO and NADPH , demonstrating the enzyme's physiological importance in controlling nitrosative stress.

Aldehyde detoxification and oxidative stress:
AKR1A1 reduces toxic aldehydes like 4-hydroxynonenal (4-HNE) that are produced during lipid peroxidation, a major consequence of oxidative stress . This function:

• Prevents 4-HNE accumulation and subsequent cellular damage

• Inhibits 4-HNE-mediated p53 activation, protecting cells from apoptosis

• Complements other detoxification systems

Studies in alcohol-fed mice demonstrate that AKR1A1 knockout results in increased oxidative stress markers, reduced antioxidant enzymes, and elevated 4-HNE levels in liver tissue .

Cross-talk with antioxidant systems:
AKR1A1 appears integrated with broader antioxidant defense mechanisms:

• Its expression may be regulated by oxidative stress-responsive transcription factors

• It requires NADPH, linking its activity to cellular redox status

• Its protective effects include maintenance of other antioxidant enzymes, as evidenced by reduced levels of these enzymes in AKR1A1-deficient mice exposed to alcohol

Tissue protection mechanisms:
AKR1A1's role in stress responses translates to protection in several tissues:

• Liver protection during chronic alcohol consumption

• Renoprotective effects during acute kidney injury through metabolic reprogramming

• Potential neuroprotection through regulation of neurotransmitter metabolism and prevention of carbonyl stress

Compensatory regulation:
A notable finding is that AKR1A1 protein levels are increased in liver extracts from GSNOR-deficient mice , suggesting compensatory upregulation when other denitrosylation systems are compromised. This indicates a degree of functional redundancy in cellular defense against nitrosative stress.

These multiple protective mechanisms position AKR1A1 as a key component of cellular defense against both oxidative and nitrosative stress, with its dysfunction potentially contributing to various pathological conditions.

What evolutionary insights can be gained from studying chicken AKR1A1 compared to other species?

Comparative analysis of chicken AKR1A1 with orthologs from other species provides valuable evolutionary insights:

Phylogenetic perspective:
AKR1A1 orthologs are found throughout the vertebrate subphylum, with the founding member identified in mammals . Birds and mammals diverged approximately 310-330 million years ago, making chicken AKR1A1 an informative model for understanding:

• Core conserved functions essential across vertebrates

• Lineage-specific adaptations in enzyme function

• Evolution of detoxification and redox regulation mechanisms

Conserved functional domains:
The core catalytic mechanisms of AKR1A1 appear remarkably conserved across species, suggesting fundamental importance in cellular metabolism. Key features likely maintained across vertebrate evolution include:

• The catalytic tetrad required for carbonyl reduction

• NADPH binding domain architecture

• Basic substrate recognition elements

Species-specific substrate preferences:
While the ability to reduce aldehydes and S-nitrosothiols is likely conserved, subtle differences in substrate preference profiles may reflect species-specific metabolic demands:

• Avian metabolism operates at higher body temperatures (40-42°C vs. 37°C in mammals)

• Birds have unique dietary and environmental exposures requiring specialized detoxification capacities

• Metabolic rates and oxygen consumption differences between species may influence needed antioxidant capacities

Structural adaptations:
Detailed structural analysis would likely reveal:

• Conservation of catalytic core residues across species

• Variable surface residues reflecting different cellular environments

• Adaptations in substrate binding pockets aligned with species-specific substrates

• Regulatory element differences that allow for species-appropriate expression patterns

Functional redundancy evolution:
The observation that mammalian AKR1A1 is upregulated in GSNOR-deficient tissues suggests evolution of compensatory mechanisms between different denitrosylation systems. Similar regulatory networks may exist in avian species, potentially with lineage-specific features.

These evolutionary insights could contribute to broader understanding of:

• The trajectory of detoxification enzyme evolution across vertebrates

• How enzymatic functions adapt to different physiological demands

• Fundamental vs. species-specific aspects of redox regulation

• Potential translational applications across species boundaries