Recombinant Herpetosiphon aurantiacus Urocanate hydratase (hutU), partial

What is Urocanate hydratase and what role does it play in histidine metabolism?

Urocanate hydratase (also known as urocanase or imidazolonepropionate hydrolase; EC 4.2.1.49) catalyzes the second step in the degradation pathway of histidine. This enzyme specifically catalyzes the hydration of urocanate to form imidazolonepropionate . In the histidine degradation pathway, histidine is first deaminated by histidine ammonia-lyase to form urocanic acid and ammonia. The urocanic acid is then converted to imidazolonepropionate by urocanate hydratase . This pathway is critical for the catabolism of histidine in many organisms, including bacteria and mammals, and represents an important metabolic route for amino acid utilization .

How do bacterial (hutU) and eukaryotic urocanate hydratase differ?

Bacterial urocanate hydratase, encoded by the hutU gene in organisms like Herpetosiphon aurantiacus, differs from the eukaryotic counterpart in several ways:

• Genetic Organization: In bacteria, the enzyme is encoded by the hutU gene, whereas in humans, it is encoded by the UROC1 gene located on chromosome 3 .

• Molecular Size: Bacterial urocanases are typically around 60 kDa, while the human enzyme is composed of 676 amino acids that fold to form a larger homodimeric structure .

• Substrate Processing: While both enzymes catalyze the same fundamental reaction, there may be differences in substrate affinity and catalytic efficiency reflecting their evolutionary divergence.

• Regulatory Control: The expression and activity of these enzymes are regulated differently in prokaryotic and eukaryotic systems, reflecting their respective metabolic contexts .

What is the clinical significance of urocanate hydratase in humans?

In humans, deficiency of urocanate hydratase leads to a condition known as urocanic aciduria, characterized by elevated levels of urocanic acid in the urine . This hereditary metabolic disorder affects the normal breakdown of histidine. Additionally, urocanic acid plays important roles beyond metabolism:

• In the epidermis, urocanic acid accumulates and may function as both a UV protectant and an immunoregulator .

• Urocanic acid exists primarily as a trans isomer (t-UA) in the uppermost layer of skin (stratum corneum), at concentrations of approximately 30 mg/cm² .

• Upon absorption of UV light, t-UA isomerizes to its cis form (c-UA), which has immunomodulatory effects including suppression of contact hypersensitivity and delayed hypersensitivity, reduction of Langerhans cell count, prolongation of skin-graft survival time, and effects on natural killer cell activity .

What are the key structural features of Urocanate hydratase?

Urocanate hydratase has several important structural features:

• It is a homodimeric enzyme in humans, composed of two identical subunits .

• The enzyme binds tightly to NAD+ which serves as an electrophilic cofactor rather than a redox factor .

• A conserved cysteine residue has been identified as crucial for the catalytic mechanism and may be involved in NAD+ binding .

• The enzyme belongs to the broader family of hydratases, containing specific domain structures consistent with other enoyl-CoA hydratases .

How does urocanate hydratase catalyze the conversion of urocanic acid?

The catalytic mechanism of urocanate hydratase involves several key steps:

• The enzyme utilizes NAD+ groups as electrophiles that attach to the top carbon of the urocanate molecule .

• This attachment triggers a sigmatropic rearrangement of the urocanate structure .

• The rearrangement facilitates the addition of a water molecule to the substrate .

• This hydration reaction converts urocanate into 4,5-dihydro-4-oxo-5-imidazolepropanoate (also called imidazolonepropionate) .

The NAD+ in this reaction does not undergo redox changes but instead serves as an electron-withdrawing group to facilitate the hydration reaction. The enzyme's active site is structured to position both the substrate and water molecule optimally for this reaction .

What are the optimal storage conditions for Recombinant Herpetosiphon aurantiacus Urocanate hydratase?

For optimal stability of Recombinant Herpetosiphon aurantiacus Urocanate hydratase, the following storage conditions are recommended:

• Short-term storage (up to one week): Store working aliquots at 4°C .

• General storage: Store at -20°C .

• Extended storage: Conserve at -20°C or -80°C .

• Avoid repeated freeze-thaw cycles as they can compromise enzyme activity and stability .

The shelf life depends on multiple factors including buffer composition, storage temperature, and the intrinsic stability of the protein. In liquid form, shelf life is typically 6 months at -20°C/-80°C, while the lyophilized form can be stable for up to 12 months at -20°C/-80°C .

What reconstitution protocol is recommended for the recombinant enzyme?

The following reconstitution protocol is recommended for optimal recovery of enzyme activity:

• Briefly centrifuge the vial before opening to bring the contents to the bottom .

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

• Add glycerol to a final concentration of 5-50% (the default recommendation is 50%) .

• Aliquot the reconstituted protein for long-term storage at -20°C/-80°C to avoid repeated freeze-thaw cycles .

This protocol helps maintain protein stability and enzymatic activity for downstream applications.

How can researchers measure urocanate hydratase activity in vitro?

Based on approaches used for similar enzymes, urocanate hydratase activity can be measured using several methods:

Coupled Enzyme Assay
A fluorescence-based coupled assay can be developed similar to those used for other hydratases :

• Primary Reaction: Urocanate hydratase converts urocanic acid to imidazolonepropionate.

• Coupling to Secondary Enzymes: The product could be coupled to a secondary enzyme reaction that produces a measurable signal.

• Detection Method: For example, by adapting the approach used for fumarate hydratase, researchers could couple the reaction to malate dehydrogenase (MDH), diaphorase, and resazurin to generate a fluorescent signal .

Direct Spectrophotometric Assay
Alternatively, direct measurement of substrate depletion or product formation may be possible:

• Urocanic acid has characteristic UV absorption properties that could be monitored during the reaction.

• Changes in absorbance at appropriate wavelengths can be measured to track the reaction progress.

This table represents an adapted protocol based on similar hydratase assays and would need optimization for hutU specifically.

What is known about the partial versus full-length Herpetosiphon aurantiacus Urocanate hydratase?

The recombinant Herpetosiphon aurantiacus Urocanate hydratase described in the search results is specifically noted as "partial" , indicating that it does not represent the full-length native protein. Key considerations regarding partial versus full-length protein include:

• Functional Domains: The partial protein likely contains the core catalytic domain necessary for enzyme function but may lack regulatory or structural regions present in the full-length protein.

• Crystallization and Structural Studies: Partial proteins are sometimes easier to crystallize for structural determination, potentially offering insights into the active site architecture.

• Stability and Solubility: Partial proteins may exhibit different stability and solubility profiles compared to full-length versions, which could affect experimental design and interpretation.

• Protein-Protein Interactions: The partial protein may lack domains involved in protein-protein interactions that could be important in the native cellular context.

Researchers using this partial recombinant protein should consider these limitations when designing experiments and interpreting results in the context of the native enzyme's function.

How do NAD+ binding dynamics affect urocanate hydratase function?

NAD+ plays a critical role in urocanate hydratase function, though not in its typical role as a redox cofactor:

• Electrophilic Catalyst: In urocanate hydratase, NAD+ acts as an electrophile rather than participating in redox reactions. It attaches to the top carbon of urocanate, triggering a sigmatropic rearrangement that facilitates water addition .

• Tight Binding: Urocanase binds NAD+ tightly, suggesting that the cofactor is an integral part of the enzyme's structure and function .

• Conserved Binding Site: A conserved cysteine residue appears to be important for the catalytic mechanism and may be involved in the binding of NAD+ .

• Structural Implications: The enzyme likely undergoes conformational changes upon NAD+ binding that position the substrate and water molecule optimally for catalysis.

Understanding these NAD+ binding dynamics is crucial for researchers investigating the catalytic mechanism of urocanate hydratase and for designing inhibitors or activators that might target this interaction.

What phylogenetic insights can be gained from studying bacterial urocanate hydratase?

Studying bacterial urocanate hydratase provides several phylogenetic insights:

• Evolutionary Conservation: Urocanate hydratase is found in bacteria (gene hutU), in the liver of many vertebrates, and has also been identified in plants such as Trifolium repens (white clover) , indicating evolutionary conservation of this metabolic pathway.

• Metabolic Adaptations: Comparative genomic studies involving Herpetosiphon aurantiacus have revealed differences in metabolic pathways among bacteria. For instance, while some Chloroflexi bacteria like Roseiflexus spp. and Chloroflexus aurantiacus contain genes for the 3-hydroxypropionate pathway for carbon fixation, Herpetosiphon aurantiacus lacks these genes .

• Functional Diversity: The presence and conservation of hutU across diverse bacterial species suggests its importance in histidine metabolism, while its absence or variation in other organisms may reflect different metabolic adaptations.

Understanding these phylogenetic relationships can provide insights into metabolic evolution and adaptation across different organisms and environments.

What experimental controls should be included in urocanate hydratase activity assays?

When designing experiments to measure urocanate hydratase activity, the following controls should be included:

Negative Controls:

• Enzyme-free control: Reaction mixture without the enzyme to establish baseline and account for non-enzymatic hydration.

• Heat-inactivated enzyme control: Denatured enzyme to verify that observed activity is due to the native protein conformation.

• Substrate-free control: Reaction mixture without urocanic acid to account for background signals.

Positive Controls:

• Reference enzyme: A well-characterized urocanate hydratase sample with known activity.

• DMSO-treated control: When testing compounds, include vehicle controls with the same DMSO concentration as in test wells .

Validation Controls:

• Counter-screen assays: When using coupled enzyme systems, include controls that directly test coupling enzyme activity (e.g., malate dehydrogenase assay) to rule out effects on these components .

• Time course measurements: Take measurements at multiple time points (e.g., 0 and 5 minutes) to ensure linear reaction rates .

These controls help ensure reliable and interpretable results by distinguishing true enzyme activity from artifacts and by providing reference points for quantification.

How can coupled enzyme assays be optimized for urocanate hydratase studies?

Optimization of coupled enzyme assays for urocanate hydratase studies requires careful consideration of several parameters:

Assay Component Optimization:

• Enzyme Concentrations: Ensure that urocanate hydratase is the rate-limiting enzyme in the coupled reaction. The coupling enzymes should be in excess to prevent them from limiting the reaction rate.

• Buffer Conditions: Optimize pH, ionic strength, and buffer composition to maintain optimal activity for all enzymes involved. For example, a buffer system similar to 50 mM Tris pH 8.0 with 5 mM MgCl₂ may be appropriate .

• Cofactor Concentrations: Ensure sufficient NAD+ for urocanate hydratase function, plus any cofactors needed for coupling enzymes.

Detection System Optimization:

• Signal-to-Noise Ratio: Adjust reagent concentrations to optimize the signal-to-noise ratio. For fluorescence-based detection using resazurin/resorufin, optimize the concentration of resazurin (e.g., 0.067 mM) for maximum sensitivity .

• Linear Range: Determine the linear range of the assay by varying substrate concentrations and measuring reaction rates. This establishes the conditions under which accurate kinetic parameters can be determined.

• Miniaturization: For high-throughput applications, optimize the assay for smaller volumes (e.g., in 1536-well plates) .

Validation Steps:

• Z'-factor Determination: Calculate the Z'-factor to assess assay quality. A value above 0.5 indicates an excellent assay for high-throughput screening.

• Counter-screens: Implement counter-screens to identify false positives or negatives. For example, a direct malate dehydrogenase counter-screen can distinguish compounds affecting urocanate hydratase from those affecting the coupling enzymes .

By systematically optimizing these parameters, researchers can develop robust and sensitive assays for studying urocanate hydratase activity in various experimental contexts.

What biophysical techniques are most informative for studying urocanate hydratase structure-function relationships?

Several biophysical techniques provide valuable insights into urocanate hydratase structure-function relationships:

Thermal Stability Assays:

• Microscale Thermophoresis (MST): This technique can assess protein stability and binding interactions by measuring changes in the movement of molecules in microscopic temperature gradients. Proteins can be labeled with fluorescent tags (such as His-Tag Labeling) to monitor these changes .

Spectroscopic Methods:

• Circular Dichroism (CD): Provides information about secondary structure elements and can monitor conformational changes upon substrate or cofactor binding.

• Fluorescence Spectroscopy: Can track changes in intrinsic tryptophan fluorescence or extrinsic fluorophores during catalysis or binding events.

Structural Determination:

• X-ray Crystallography: Provides high-resolution structural information, especially valuable for understanding the active site architecture and substrate binding mode.

• Cryo-Electron Microscopy: Can reveal structural features of larger complexes or conformational states that may be difficult to crystallize.

Binding Studies:

• Isothermal Titration Calorimetry (ITC): Measures the thermodynamic parameters of binding interactions, providing insights into the energetics of substrate and cofactor binding.

• Surface Plasmon Resonance (SPR): Offers real-time monitoring of binding kinetics between the enzyme and its substrates, products, or potential inhibitors.

Computational Approaches:

• Molecular Dynamics Simulations: Can model enzyme dynamics and conformational changes during the catalytic cycle.

• Quantum Mechanics/Molecular Mechanics (QM/MM): Particularly useful for understanding the electronic aspects of the catalytic mechanism involving NAD+ as an electrophilic cofactor.

By integrating data from multiple biophysical techniques, researchers can develop comprehensive models of urocanate hydratase structure-function relationships, informing both basic understanding and applied research such as inhibitor design.

What are common challenges in interpreting urocanate hydratase kinetic data?

Researchers analyzing urocanate hydratase kinetic data should be aware of several common challenges:

• NAD+ Binding Complexity: Since NAD+ acts as an electrophilic cofactor rather than a redox cofactor in urocanate hydratase , standard Michaelis-Menten kinetics may not fully describe the enzyme behavior. The tight binding of NAD+ may complicate kinetic analysis, requiring more complex models.

• Coupled Assay Interference: When using coupled enzyme assays, compounds being tested might affect the coupling enzymes rather than urocanate hydratase itself. This necessitates careful counter-screening, as described for similar hydratase assays .

• Product Inhibition: Imidazolonepropionate may exhibit product inhibition, affecting reaction rates at higher concentrations or longer reaction times. Time-course measurements can help identify this effect.

• Allosteric Effects: Urocanate hydratase may exhibit allosteric regulation, particularly if it functions as a homodimer . This could lead to non-linear kinetics that require more sophisticated modeling approaches.

• Partial Protein Limitations: When working with a partial recombinant protein , certain regulatory domains may be missing, potentially altering kinetic behavior compared to the full-length enzyme.

Addressing these challenges requires careful experimental design, appropriate controls, and sophisticated data analysis approaches to accurately characterize urocanate hydratase kinetics.

How should researchers analyze structure-function data for urocanate hydratase?

Effective analysis of structure-function data for urocanate hydratase involves several key approaches:

• Integrative Structural Analysis: Combine data from multiple structural techniques (X-ray crystallography, CD spectroscopy, etc.) to develop a comprehensive structural model. Pay particular attention to the NAD+ binding site and the conserved cysteine residue implicated in catalysis .

• Correlation of Structural Features with Function:

  • Map conserved residues onto the structural model to identify functionally important regions.

  • Analyze the structural basis for homodimer formation and assess its importance for function .

  • Examine structural changes upon substrate binding and during catalysis.

• Mutagenesis Data Interpretation:

  • Create a structure-based rationalization for the effects of mutations, particularly those affecting the conserved cysteine residue .

  • Use site-directed mutagenesis data to validate structural hypotheses about the catalytic mechanism.

• Molecular Dynamics Interpretation:

  • Analyze trajectories for conformational changes related to substrate binding and catalysis.

  • Identify water molecules involved in the hydration reaction and their positioning within the active site.

• Comparative Analysis:

  • Compare structural features of bacterial hutU with eukaryotic urocanases to identify conserved and divergent elements.

  • Use structural comparisons with related hydratases to identify common mechanistic themes.

By systematically integrating structural and functional data, researchers can develop mechanistic models that explain how urocanate hydratase's structure enables its specific catalytic function in histidine metabolism.

What are promising applications of urocanate hydratase in biotechnology?

Several promising biotechnological applications for urocanate hydratase warrant investigation:

• Biosensors for Histidine and Derivatives:

  • Development of enzyme-based biosensors for detecting histidine levels in biological samples.

  • Potential applications in medical diagnostics, particularly for conditions associated with histidine metabolism disorders.

• Biocatalysis for Chemical Synthesis:

  • Utilizing urocanate hydratase for stereoselective hydration reactions in the synthesis of specialized chemicals.

  • Potential for creating novel imidazole derivatives with pharmaceutical applications.

• Environmental Monitoring:

  • Development of systems to detect histidine and related compounds in environmental samples.

  • Potential application in monitoring microbial activity in various ecosystems.

• Protein Engineering:

  • Engineering urocanate hydratase variants with altered substrate specificity or improved stability.

  • Creating chimeric enzymes combining domains from different hydratases to develop novel catalytic functions.

• Therapeutic Applications:

  • Development of modulators of urocanate hydratase activity for potential treatment of urocanic aciduria .

  • Investigation of the enzyme's role in UV protection and immunomodulation for potential dermatological applications .

These applications represent opportunities to translate fundamental understanding of urocanate hydratase into practical biotechnological tools and therapeutic strategies.

What key questions remain unanswered about bacterial urocanate hydratase?

Despite current knowledge, several important questions about bacterial urocanate hydratase remain unanswered:

• Evolutionary Significance:

  • Why do some bacteria like Herpetosiphon aurantiacus maintain the hutU gene while lacking other related metabolic pathways found in related species ?

  • What selective pressures have shaped the evolution of bacterial urocanate hydratases?

• Regulatory Mechanisms:

  • How is hutU expression regulated in bacterial systems in response to environmental conditions?

  • Are there allosteric regulators of urocanate hydratase activity in bacteria?

• Structural Determinants of Function:

  • What specific structural features distinguish bacterial hutU from eukaryotic urocanases?

  • How do these structural differences relate to functional differences?

• Ecological Role:

  • What is the ecological significance of histidine metabolism via urocanate hydratase in bacterial communities?

  • How does this pathway interact with other metabolic networks in different environments?

• Substrate Promiscuity:

  • Can bacterial urocanate hydratase act on substrates other than urocanic acid?

  • What structural features determine substrate specificity?

Addressing these questions will require interdisciplinary approaches combining structural biology, biochemistry, genetics, and ecological studies, potentially yielding new insights into both basic microbial physiology and potential biotechnological applications.

What are best practices for working with Recombinant Herpetosiphon aurantiacus Urocanate hydratase?

Based on the available information, researchers working with Recombinant Herpetosiphon aurantiacus Urocanate hydratase should follow these best practices:

• Storage and Handling:

  • Store the enzyme at -20°C for routine use, or at -80°C for extended storage .

  • Prepare working aliquots to avoid repeated freeze-thaw cycles, which can compromise activity .

  • For short-term work (up to one week), working aliquots can be stored at 4°C .

• Reconstitution:

  • Centrifuge the vial briefly before opening to collect the product at the bottom .

  • Reconstitute in deionized sterile water to 0.1-1.0 mg/mL .

  • Add glycerol to a final concentration of 5-50% (50% recommended) for stability .

  • Prepare multiple small aliquots for long-term storage to avoid repeated freeze-thaw cycles .

• Activity Assays:

  • Consider coupled enzyme assays similar to those used for other hydratases .

  • Include appropriate controls for enzyme activity, substrate specificity, and assay validation.

  • Monitor reaction progress at multiple time points to ensure linearity.

• Data Interpretation:

  • Remember that you are working with a partial protein , which may have different properties than the full-length enzyme.

  • Consider the role of NAD+ as an electrophilic cofactor rather than a redox cofactor .

  • Account for the homodimeric nature of the native enzyme when interpreting results .

Following these practices will help ensure reliable and reproducible results when working with this recombinant enzyme.

What complementary techniques should researchers consider when studying urocanate hydratase?

To gain comprehensive insights into urocanate hydratase function and mechanisms, researchers should consider these complementary techniques:

• Enzymatic Characterization:

  • Steady-state kinetics to determine KM, kcat, and catalytic efficiency

  • Pre-steady-state kinetics to identify rate-limiting steps

  • pH-rate profiles to identify key ionizable groups in catalysis

  • Temperature dependence studies to determine activation parameters

• Structural Characterization:

  • X-ray crystallography or cryo-EM for high-resolution structural determination

  • Small-angle X-ray scattering (SAXS) for solution-state structural information

  • Hydrogen-deuterium exchange mass spectrometry to probe conformational dynamics

• Binding Studies:

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters of binding

  • Microscale thermophoresis for binding affinity determination

  • Surface plasmon resonance for binding kinetics

• Molecular Biology Approaches:

  • Site-directed mutagenesis to probe the role of specific residues

  • Gene expression analysis to understand regulation

  • Protein engineering to create variants with altered properties

• Computational Methods:

  • Molecular dynamics simulations to study enzyme dynamics

  • Quantum mechanics/molecular mechanics (QM/MM) to investigate the reaction mechanism

  • Bioinformatic analyses for evolutionary relationships and conserved features