Recombinant Shewanella halifaxensis Urocanate hydratase (hutU), partial

What is Urocanate hydratase (hutU) and what is its function in histidine metabolism?

Urocanate hydratase (hutU) is a key enzyme in the histidine utilization (Hut) pathway, catalyzing the conversion of urocanate to imidazolonepropionate. Also known as urocanase or imidazolonepropionate hydrolase (EC 4.2.1.49), it plays a critical role in allowing bacteria to utilize histidine as a carbon and nitrogen source . This enzyme differs from urocanate reductase (UrdA), which catalyzes the reduction of urocanic acid to deamino-histidine through a two-electron reduction process .

The reaction mechanism involves hydration of the carbon-nitrogen double bond in urocanate, representing a critical step in the degradation pathway that eventually feeds into central metabolism. Understanding this enzyme's function provides insights into how Shewanella species metabolize nitrogen-containing compounds.

Why is Shewanella halifaxensis significant in environmental microbiology?

Shewanella halifaxensis is a marine bacterium with unique metabolic capabilities that make it significant for environmental research. This obligately respiratory, denitrifying bacterium was isolated from marine sediment in the Emerald Basin (215 m depth) of the Atlantic Ocean near Halifax, Canada . Its ecological importance stems from several notable characteristics:

• Ability to degrade explosive compounds such as hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)

• Demonstrated algicidal activity against harmful algal blooms (HABs), particularly against the dinoflagellate Prorocentrum triestinum

• Metabolic versatility, capable of utilizing various carbon sources including peptone, yeast extract, Casamino acids, esters, sugars, and amino acids like serine and proline

These properties position S. halifaxensis as a promising candidate for bioremediation applications and biological control of harmful algal blooms, making its enzymes, including hutU, valuable targets for research.

How does Shewanella halifaxensis relate to other Shewanella species?

Shewanella halifaxensis belongs to the genus Shewanella within the family Shewanellaceae and order Alteromonadales. Phylogenetic analysis based on 16S rRNA sequences has shown that S. halifaxensis forms a distinct clade with high conservation (bootstrap score over 75) . When compared to its closest relative, Shewanella pealeana, DNA-DNA hybridization revealed only 17.9% relatedness, well below the 70% species cut-off value, confirming its status as a distinct species .

What are the optimal conditions for expressing recombinant S. halifaxensis hutU?

Based on experimental approaches with similar enzymes, the following conditions are recommended for optimal expression of recombinant S. halifaxensis hutU:

When purifying the enzyme, consider that as a deep-sea bacterium adapted to cold environments, its enzymes may exhibit optimal activity and stability at lower temperatures than typical mesophilic bacteria. The recombinant protein should be stored at -20°C/-80°C, with lyophilized forms typically maintaining shelf life for up to 12 months and liquid forms for approximately 6 months .

How can enzymatic activity of S. halifaxensis hutU be accurately measured?

Accurate measurement of hutU enzymatic activity can be accomplished through several complementary approaches:

• Spectrophotometric Assay:

  • Monitor the decrease in absorbance at 277 nm, which corresponds to the consumption of urocanate

  • Reaction buffer should mimic marine conditions with appropriate salinity

  • Include controls to account for non-enzymatic hydration

• HPLC Analysis:

  • Quantify both substrate depletion and product formation

  • Use reverse-phase HPLC with UV detection at 210-220 nm for imidazolonepropionate

  • Compare against standardized curves of both substrate and product

• Enzyme Kinetics Determination:

  • Calculate Km and kcat using varied substrate concentrations

  • For related enzymes like urocanate reductase, kinetic parameters include high substrate affinity (Km << 10 μM) and high turnover rate (kcat = 360 s-1)

  • Determine pH optimum, likely in the range of 7.0-8.0 to reflect marine environment

When designing these assays, it's essential to consider the potential impact of temperature, as S. halifaxensis is adapted to deep-sea environments with consistently cold temperatures .

What purification strategies are most effective for recombinant S. halifaxensis hutU?

A multi-step purification strategy is recommended for obtaining high-purity recombinant S. halifaxensis hutU:

Throughout purification, maintain cold conditions (4°C) to preserve enzyme activity, especially given S. halifaxensis' adaptation to cold environments. Buffer systems should include stabilizing agents such as glycerol (5-10%) and potentially reducing agents to protect any critical cysteine residues .

After purification, reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL with 5-50% glycerol for long-term storage at -20°C/-80°C .

How might S. halifaxensis hutU contribute to environmental bioremediation applications?

S. halifaxensis hutU's potential in bioremediation stems from this bacterium's unique metabolic capabilities:

The development of immobilized enzyme systems or engineered bacterial strains with enhanced hutU activity could significantly advance these bioremediation applications.

What implications does hutU research have for understanding anaerobic respiration in Shewanella species?

Research on hutU in S. halifaxensis has significant implications for understanding alternative respiratory pathways in Shewanella species:

• Novel Electron Acceptor Pathways:

  • Related research has identified that Shewanella oneidensis MR-1 utilizes urocanate reductase (UrdA) in a novel anaerobic respiratory pathway where urocanic acid serves as a terminal electron acceptor

  • This pathway enables growth in environments where no other bacteria can compete

  • Understanding hutU's role in urocanic acid metabolism could reveal interactions with these alternative respiratory pathways

• Metabolic Integration:

  • As S. halifaxensis is described as an "obligately respiratory, denitrifying" bacterium , there may be connections between histidine metabolism via hutU and denitrification pathways

  • These connections could explain how Shewanella species adapt to various environmental conditions through metabolic flexibility

• Evolutionary Adaptations:

  • The presence of both hutU (hydratase) and UrdA (reductase) acting on the same substrate (urocanic acid) but catalyzing different reactions suggests sophisticated evolutionary tuning of metabolic pathways

  • This adaptation likely enables Shewanella species to thrive in diverse ecological niches including deep-sea environments

Comparative analysis of hutU and UrdA could provide insights into how these enzymes have evolved to support different metabolic strategies within the Shewanella genus.

How can advanced structural biology techniques enhance our understanding of S. halifaxensis hutU?

Advanced structural biology techniques can provide crucial insights into S. halifaxensis hutU's function and catalytic mechanism:

• X-ray Crystallography and Cryo-EM:

  • Determine the three-dimensional structure at atomic resolution

  • Identify catalytic residues and substrate-binding pocket architecture

  • Compare with structures of related enzymes like urocanate reductase for evolutionary insights

  • Structural data would complement the sequence information available for related urocanate hydratases

• Molecular Dynamics Simulations:

  • Model enzyme flexibility and conformational changes during catalysis

  • Simulate the effects of marine conditions (pressure, salinity) on enzyme structure

  • Investigate how cold adaptation features are reflected in protein dynamics

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Map protein-substrate interactions and conformational changes

  • Identify regions with different solvent accessibility during catalysis

  • Provide insights into enzyme dynamics without requiring crystallization

• Structure-Guided Protein Engineering:

  • Design variants with enhanced stability or altered substrate specificity

  • Engineer cold-active variants that maintain activity at even lower temperatures

  • Develop mutants with potential applications in bioremediation

These approaches would be particularly valuable for understanding how hutU from a deep-sea bacterium like S. halifaxensis has adapted to function in cold, high-pressure environments .

What cofactors and critical residues are essential for S. halifaxensis hutU activity?

While specific information on S. halifaxensis hutU cofactors is not directly reported in the literature, comparative analysis with related enzymes suggests the following:

• Potential Cofactors:

  • Unlike urocanate reductase (UrdA), which requires flavin as a cofactor , urocanate hydratase typically does not require redox cofactors for its hydration reaction

  • Metal ions (particularly divalent cations like Mg2+ or Mn2+) may play a role in stabilizing the transition state during catalysis

  • The enzyme likely contains a water molecule in the active site that participates directly in the hydration reaction

• Critical Catalytic Residues:

  • Conserved acidic residues (Asp, Glu) likely function as general base catalysts, activating water for nucleophilic attack on urocanate

  • Basic residues (Lys, Arg, His) may coordinate the carboxylate group of urocanate and stabilize negative charge development during the reaction

  • Comparative sequence analysis with related hydratases would identify these conserved residues

• Substrate-Binding Pocket:

  • Aromatic residues likely participate in π-stacking interactions with the imidazole ring of urocanate

  • Hydrogen-bonding residues coordinate the carboxylate group and imidazole nitrogen

Mutagenesis studies targeting these predicted critical residues would confirm their roles in catalysis and substrate binding.

How does temperature affect the stability and activity of S. halifaxensis hutU?

As S. halifaxensis is a deep-sea bacterium isolated from the Atlantic Ocean at a depth of 215 m , its enzymes, including hutU, are likely to display cold-adaptation features:

• Temperature-Activity Profile:

  • Expected temperature optimum around 10-15°C, consistent with S. halifaxensis' optimal growth at 10°C

  • Higher catalytic efficiency (kcat/Km) at low temperatures compared to mesophilic homologs

  • Activity may decrease rapidly above 25-30°C due to conformational flexibility

• Thermal Stability Characteristics:

  • Lower thermal stability compared to mesophilic enzymes

  • Partially compensated by increased structural flexibility at low temperatures

  • Storage stability likely enhanced at -20°C/-80°C, with lyophilized forms maintaining activity for up to 12 months

• Structural Adaptations:

  • Fewer ionic interactions and hydrogen bonds, allowing greater flexibility at low temperatures

  • Potential reduction in proline content in loop regions

  • Modified surface charge distribution to maintain solubility in cold environments

These temperature-related characteristics would be critical considerations when designing experimental protocols for recombinant S. halifaxensis hutU.

What substrate specificity profile does S. halifaxensis hutU exhibit?

The substrate specificity of S. halifaxensis hutU would be characterized by:

• Primary Substrate:

  • Urocanate (trans-urocanic acid) is expected to be the primary physiological substrate

  • High specificity similar to that observed for urocanate reductase in S. oneidensis, which shows strong specificity for urocanic acid

• Potential Secondary Substrates:

  • Structurally similar α,β-unsaturated carboxylic acids with nitrogen-containing aromatic rings

  • Histidine derivatives with modifications that preserve the imidazole acrylic acid structure

• Specificity Determinants:

  • Recognition likely depends on both the imidazole ring and the acrylic acid moiety

  • The trans configuration of the double bond is expected to be critical for proper binding

A comprehensive substrate specificity profile would involve testing:

Kinetic analysis with these compounds would provide valuable insights into the structural requirements for substrate recognition.

What expression systems are optimal for producing active recombinant S. halifaxensis hutU?

Several expression systems could be considered for recombinant S. halifaxensis hutU production, each with distinct advantages:

• E. coli Expression Systems:

  • BL21(DE3): Standard system for initial expression trials

  • Arctic Express: Contains cold-adapted chaperonins, beneficial for expressing proteins from psychrophilic organisms like S. halifaxensis

  • Rosetta: Provides rare codons that may be present in S. halifaxensis genes

  • SHuffle: Enhanced disulfide bond formation if hutU contains structural disulfides

• Yeast Expression Systems:

  • Pichia pastoris: Effective for secreted expression with proper folding

  • Benefits from eukaryotic post-translational modification machinery

  • Can achieve high cell densities in bioreactors

• Insect Cell Systems:

  • Baculovirus expression system (used successfully for similar enzymes)

  • Excellent for complex proteins requiring specific folding conditions

  • More expensive but potentially higher yield of correctly folded protein

• Cell-Free Expression Systems:

  • Rapid screening of constructs and conditions

  • Allows incorporation of unnatural amino acids for mechanistic studies

  • Avoids potential toxicity issues

The optimal choice depends on specific research goals and downstream applications. For structural studies requiring large quantities of pure protein, baculovirus or Pichia systems may be preferred, while E. coli systems offer simplicity and cost-effectiveness for initial characterization.

How can site-directed mutagenesis be applied to enhance S. halifaxensis hutU stability or activity?

Site-directed mutagenesis represents a powerful approach for enhancing S. halifaxensis hutU properties:

• Stability Enhancement Strategies:

  • Introduce additional salt bridges or disulfide bonds to enhance thermal stability

  • Replace thermolabile residues (Asn, Gln) in critical positions

  • Engineer surface residues to reduce aggregation propensity

  • Add proline residues in loop regions to reduce flexibility at higher temperatures

• Activity Enhancement Approaches:

  • Modify substrate binding pocket residues to improve affinity (lower Km)

  • Target catalytic residues to enhance turnover rate (higher kcat)

  • Engineer the active site entrance to improve substrate access and product release

  • Introduce mutations that reduce product inhibition

• Systematic Mutagenesis Workflow:

For a cold-adapted enzyme like S. halifaxensis hutU, a particular focus might be engineering variants that maintain cold activity while increasing thermal stability for broader application conditions.

What strategies can optimize codon usage for heterologous expression of S. halifaxensis hutU?

Optimizing codon usage is critical for efficient heterologous expression of S. halifaxensis hutU:

• Codon Optimization Principles:

  • Adapt codons to match preference in expression host (E. coli, yeast, insect cells)

  • Balance GC content to improve mRNA stability and translation efficiency

  • Remove rare codons that could cause translational pausing

  • Eliminate potential cryptic splice sites if expressing in eukaryotic systems

• Marine Bacteria-Specific Considerations:

  • S. halifaxensis, as a deep-sea bacterium , may have codon usage patterns adapted to cold environments

  • Its native GC content (45 mol%) should be considered when designing synthetic genes

  • Potential secondary structure formation in the mRNA should be minimized

• Expression Enhancement Elements:

  • Optimize the 5' untranslated region to enhance ribosome binding

  • Consider adding translation enhancing sequences (TES)

  • Engineer optimal distance between Shine-Dalgarno sequence and start codon in bacterial systems

• Software Tools and Resources:

  • Specialized algorithms that consider both codon preference and mRNA structure

  • Codon optimization tools should be selected based on the specific expression host

  • Custom synthesis of the optimized gene is often more cost-effective than extensive cloning efforts

These optimization strategies can significantly improve expression yields, particularly for proteins from organisms with divergent codon usage patterns like marine bacteria.

How does hutU integrate with other metabolic pathways in S. halifaxensis?

Urocanate hydratase (hutU) in S. halifaxensis likely integrates with multiple metabolic pathways:

• Histidine Utilization Pathway:

  • Functions in the canonical histidine degradation pathway:
    Histidine → Urocanate → Imidazolonepropionate → Formiminoglutamate → Glutamate

  • Enables utilization of histidine as both carbon and nitrogen source

  • Connected to glutamate metabolism, feeding into central carbon metabolism

• Connection to Nitrogen Metabolism:

  • S. halifaxensis is described as a denitrifying bacterium

  • Nitrogen from histidine degradation may be redirected into various nitrogen-containing compounds

  • May interface with specialized pathways for explosive compound degradation

• Relationship to Anaerobic Respiration:

  • In the related organism S. oneidensis, urocanic acid serves as a terminal electron acceptor via urocanate reductase (UrdA)

  • HutU and UrdA may represent competing pathways for urocanic acid utilization under different conditions

  • The balance between these pathways could be regulated by oxygen availability

• Potential Role in Algicidal Activity:

  • S. halifaxensis produces bioactive compounds with algicidal activity

  • Histidine metabolism might provide precursors for these specialized metabolites

  • Understanding hutU's role could provide insights into the production of these compounds

A systems biology approach combining transcriptomics, proteomics, and metabolomics would be necessary to fully characterize these metabolic connections.

What omics approaches would best elucidate the role of hutU in S. halifaxensis metabolism?

Multiple omics approaches can provide complementary insights into hutU's role:

• Transcriptomics:

  • RNA-Seq to identify genes co-expressed with hutU under various conditions

  • Comparison between aerobic and anaerobic conditions to understand regulation

  • Analysis of expression patterns during growth on different nitrogen sources

  • Identification of potential regulatory elements controlling hutU expression

• Proteomics:

  • Quantitative proteomics to measure enzyme abundance across conditions

  • Protein-protein interaction studies to identify complexes involving hutU

  • Post-translational modification analysis to understand regulation

  • Targeted proteomics focusing on histidine metabolism enzymes

• Metabolomics:

  • Targeted analysis of histidine pathway intermediates

  • Stable isotope labeling to trace carbon and nitrogen flux

  • Untargeted metabolomics to identify novel connections to other pathways

  • Comparative analysis between wild-type and hutU knockout strains

• Integrated Multi-omics Approach:

This comprehensive approach would provide a systems-level understanding of how hutU contributes to S. halifaxensis' unique metabolic capabilities.

How might environmental factors influence hutU expression and activity in S. halifaxensis?

As a deep-sea bacterium, S. halifaxensis experiences a unique set of environmental conditions that likely influence hutU expression and activity:

• Temperature Effects:

  • Native habitat at 215 m depth in the Atlantic Ocean suggests adaptation to cold, relatively stable temperatures

  • hutU expression may be regulated by temperature-sensitive transcription factors

  • Enzyme activity likely optimized for consistent cold temperatures rather than temperature fluctuations

• Oxygen Availability:

  • S. halifaxensis is described as an "obligately respiratory, denitrifying" bacterium

  • hutU expression may be differentially regulated under aerobic versus anaerobic conditions

  • Related research shows that urocanate reductase in S. oneidensis is induced under anaerobic conditions

• Nutrient Availability:

  • Expression likely responsive to histidine availability

  • May be coordinated with other amino acid utilization pathways

  • S. halifaxensis can utilize various carbon sources including amino acids , suggesting sophisticated metabolic regulation

• Marine Environment Factors:

  • Salinity may affect enzyme structure and function

  • Hydrostatic pressure at depth could influence protein folding and activity

  • Potential coordination with algicidal compound production under specific environmental cues

Experimental approaches to study these effects would include gene expression analysis under varied conditions and enzymatic characterization across relevant environmental parameters.

What are the most promising applications for engineered variants of S. halifaxensis hutU?

Engineered variants of S. halifaxensis hutU offer several promising applications:

• Bioremediation Technologies:

  • Enhanced variants could improve S. halifaxensis' ability to degrade explosive compounds

  • Immobilized enzyme systems for environmental cleanup of contaminated marine environments

  • Integration into bioreactors for continuous treatment processes

• Control of Harmful Algal Blooms:

  • If hutU metabolic products contribute to S. halifaxensis' algicidal activity , engineered variants could enhance this effect

  • Development of enzyme-based treatments with improved stability across temperature and pH ranges

  • Targeted approaches to specific harmful dinoflagellate species

• Biotechnological Applications:

  • Cold-active enzymes for industrial processes requiring low-temperature reactions

  • Novel biocatalysts for stereospecific hydration reactions in pharmaceutical synthesis

  • Biosensors for histidine or urocanate detection in environmental samples

• Fundamental Research Tools:

  • Engineered variants with improved stability for structural studies

  • Reporter constructs for studying histidine metabolism in various environments

  • Model systems for studying cold adaptation in enzymes

These applications leverage S. halifaxensis hutU's unique properties as an enzyme from a marine bacterium with specialized metabolic capabilities.

What unresolved questions remain about the catalytic mechanism of hutU enzymes?

Several critical questions remain unresolved regarding hutU's catalytic mechanism:

• Reaction Stereochemistry:

  • Is the hydration reaction stereospecific?

  • What determines the stereochemical outcome?

  • How does the enzyme control the addition of water to the double bond?

• Catalytic Residues:

  • Which specific residues activate the water molecule for nucleophilic attack?

  • What residues coordinate and position the substrate?

  • How does the enzyme stabilize the transition state?

• Conformational Changes:

  • Do significant domain movements occur during catalysis?

  • Is there an induced fit mechanism upon substrate binding?

  • How does product release influence the catalytic cycle?

• Species-Specific Adaptations:

  • How has the catalytic mechanism evolved in deep-sea bacteria like S. halifaxensis?

  • Are there unique features that distinguish hutU from S. halifaxensis from homologs in mesophilic bacteria?

  • What structural adaptations enable function in cold, high-pressure environments?

Resolving these questions would require integrated approaches combining structural biology, enzyme kinetics, and computational modeling.

How might CRISPR-Cas9 genome editing advance our understanding of hutU function in S. halifaxensis?

CRISPR-Cas9 genome editing offers powerful approaches to study hutU function in S. halifaxensis:

• Precise Gene Manipulation:

  • Knockout studies to determine hutU essentiality under various growth conditions

  • Introduction of point mutations to test hypotheses about catalytic residues

  • Domain swapping with homologs from other species to identify adaptation-specific regions

  • Reporter gene fusions to monitor expression in real-time

• Regulatory Studies:

  • Modification of promoter regions to understand transcriptional control

  • Targeting of potential transcription factors to elucidate regulatory networks

  • Engineering of inducible systems for controlled expression

• Metabolic Engineering:

  • Integration of hutU with other metabolic pathways to enhance desired capabilities

  • Modification of the histidine utilization pathway to improve explosive compound degradation

  • Engineering enhanced production of algicidal compounds

• Systematic Genome-Wide Studies:

  • CRISPR interference (CRISPRi) screens to identify genetic interactions

  • Creation of variant libraries to support directed evolution approaches

  • Integration with omics data for systems-level understanding

These approaches would significantly advance our understanding of hutU's role in S. halifaxensis' unique metabolic capabilities and ecological adaptations.