Recombinant Ochrobactrum anthropi Urocanate hydratase (hutU), partial

What is Ochrobactrum anthropi and why is it significant in research?

Ochrobactrum anthropi is a gram-negative, obligately aerobic, non-lactose-fermenting bacillus that is widely distributed in the environment, including soil, plants, and water sources. While it was formerly classified as CDC group Vd, it is now recognized as belonging to the genus Ochrobactrum, which is closely related to Brucella species . O. anthropi has gained research significance as an emerging opportunistic pathogen that can cause infections in both immunocompetent and immunocompromised individuals, including bacteremia and localized infections such as osteomyelitis . The organism has intrinsic resistance to multiple antibiotics, particularly β-lactams, making it clinically relevant in the study of antimicrobial resistance mechanisms .

What is urocanate hydratase (hutU) and what is its role in bacterial metabolism?

Urocanate hydratase (hutU) is an enzyme involved in the histidine utilization (hut) pathway in bacteria. This enzyme catalyzes the conversion of urocanate to 4-imidazolone-5-propionate in the second step of histidine catabolism. In bacterial systems like O. anthropi, this pathway allows the organism to use histidine as a carbon and nitrogen source, particularly in nutrient-limited environments. The study of hutU contributes to our understanding of bacterial adaptability and metabolic diversity, particularly in organisms that can survive in diverse ecological niches.

How does recombinant partial hutU differ from the native enzyme?

Recombinant partial hutU refers to a fragment of the full hutU enzyme that has been produced through genetic engineering techniques. The partial nature of the recombinant protein may reflect challenges in expressing the complete enzyme or strategic decisions to focus on specific functional domains. When comparing recombinant partial hutU to the native enzyme, researchers should consider potential differences in catalytic efficiency, structural integrity, and stability. The partial recombinant version may lack certain regulatory domains or may exhibit altered kinetic properties compared to the native enzyme isolated directly from O. anthropi.

What are the optimal expression systems for recombinant O. anthropi hutU?

For recombinant expression of O. anthropi hutU, E. coli-based expression systems are commonly employed due to their high yield and ease of genetic manipulation. When selecting an expression system, researchers should consider:

• Expression vectors with strong inducible promoters (T7, tac, or araBAD)

• E. coli strains optimized for recombinant protein expression (BL21(DE3), Rosetta, or Origami for proteins requiring disulfide bonds)

• Addition of fusion tags (His6, GST, or MBP) to facilitate purification and potentially improve solubility

• Codon optimization based on O. anthropi's distinct codon usage patterns

For optimal expression, induction conditions should be empirically determined, testing various temperatures (16-37°C), inducer concentrations, and induction durations to balance protein yield with proper folding and solubility.

What purification strategies yield highest purity and activity for recombinant hutU?

A multi-step purification protocol is recommended to achieve high purity while maintaining enzymatic activity:

• Initial capture: Immobilized metal affinity chromatography (IMAC) for His-tagged constructs or glutathione affinity chromatography for GST-fusion proteins

• Intermediate purification: Ion exchange chromatography (typically anion exchange at pH 7.5-8.0)

• Polishing step: Size exclusion chromatography to remove aggregates and achieve homogeneity

Throughout purification, it's critical to monitor enzyme activity with a spectrophotometric assay measuring the decrease in absorbance at 277 nm as urocanate is converted to 4-imidazolone-5-propionate. Buffer optimization should include stability screening with various pH conditions (typically pH 7.0-8.5) and potential stabilizing additives such as glycerol (10-20%) or reducing agents like DTT or β-mercaptoethanol if cysteine residues are present.

How can researchers troubleshoot poor expression or insolubility issues?

When encountering expression or solubility challenges with recombinant hutU, consider implementing these stepwise troubleshooting strategies:

• For poor expression:

  • Verify sequence integrity and codon optimization

  • Test alternative promoters or expression hosts

  • Evaluate media composition and growth conditions

  • Implement auto-induction media or cell-free expression systems

• For insolubility issues:

  • Lower induction temperature (16-20°C) and inducer concentration

  • Co-express with molecular chaperones (GroEL/ES, DnaK/J)

  • Employ solubility-enhancing fusion partners (MBP, SUMO, or Trx)

  • Test refolding protocols if inclusion bodies persist

  • Explore detergent screening if membrane association is suspected

Systematic documentation of each optimization attempt is essential for identifying the critical parameters affecting hutU expression and solubility.

What assays are most effective for measuring hutU enzymatic activity?

The standard assay for urocanate hydratase activity involves spectrophotometric monitoring of urocanate conversion at 277 nm (ε = 18,800 M⁻¹ cm⁻¹). A typical reaction mixture contains:

• 50 mM phosphate buffer (pH 7.5)

• 0.1 mM urocanate

• Purified enzyme (0.1-1 μg)

• Total volume: 1 ml

Activity is typically measured at 25°C with continuous monitoring for 5 minutes. One unit of enzyme activity is defined as the amount of enzyme that catalyzes the conversion of 1 μmol of urocanate per minute under the assay conditions.

For more sensitive detection, HPLC-based methods can be employed to simultaneously monitor substrate depletion and product formation, particularly useful when analyzing complex biological samples or when substrate/product have overlapping spectral properties.

What are the kinetic parameters of recombinant O. anthropi hutU?

While specific kinetic parameters for O. anthropi hutU must be determined experimentally, typical values for bacterial urocanate hydratases include:

Researchers should systematically evaluate these parameters for the specific recombinant partial hutU preparation, as they may differ from other bacterial sources due to sequence variations or the partial nature of the recombinant construct.

How does the enzyme stability vary under different storage and reaction conditions?

Storage stability and reaction condition optimization are critical for maintaining hutU activity. Based on studies with similar enzymes:

• Short-term storage (1-2 weeks):

  • 4°C in buffer containing 50 mM phosphate (pH 7.5), 150 mM NaCl, 1 mM DTT, and 10% glycerol

• Long-term storage:

  • -80°C in the same buffer with 20-25% glycerol

  • Lyophilization may be considered after buffer exchange to remove salts

• Critical stability factors:

  • Temperature: Significant activity loss typically occurs above 40°C

  • pH: Stability generally decreases below pH 6.0 and above pH 9.0

  • Metal ions: Divalent cations (Mg²⁺, Mn²⁺) at 1-5 mM may enhance stability

  • Reducing agents: DTT or β-mercaptoethanol (1-5 mM) may prevent oxidative inactivation

Half-life determinations under various conditions should be performed to establish optimal handling protocols for specific research applications.

What structural features distinguish O. anthropi hutU from other bacterial urocanate hydratases?

O. anthropi hutU belongs to the α/β barrel fold family characteristic of many urocanate hydratases. While specific structural information for O. anthropi hutU requires experimental determination through X-ray crystallography or cryo-EM, comparative sequence analysis suggests:

• The catalytic domain likely contains conserved residues for substrate binding, including histidine and aspartate residues that coordinate with the imidazole ring of urocanate

• The enzyme may exhibit sequence divergence in loop regions that could influence substrate specificity or regulatory interactions

• Like other urocanate hydratases, it may contain metal-binding sites, potentially involving zinc or iron as cofactors

Homology modeling based on related bacterial urocanate hydratases could provide preliminary structural insights prior to experimental structure determination.

How can site-directed mutagenesis be used to study functional domains in hutU?

Site-directed mutagenesis represents a powerful approach for investigating structure-function relationships in hutU. A systematic mutagenesis strategy should target:

• Putative catalytic residues identified through sequence alignment with characterized urocanate hydratases

• Metal-binding sites if cofactor dependence is observed

• Substrate-binding pocket residues that may confer specificity

• Regions unique to O. anthropi hutU compared to other bacterial homologs

Each mutant should be characterized using:

• Enzyme activity assays to determine kinetic consequences (k_cat, K_m)

• Thermal stability assessments (differential scanning fluorimetry)

• Substrate specificity profiles with structural analogs

• Cofactor binding studies if applicable

The combined data from multiple mutants can help construct a functional map of the enzyme's active site and regulatory domains.

What advanced techniques can reveal the catalytic mechanism of hutU?

Understanding the catalytic mechanism of hutU requires integrating multiple biophysical approaches:

• Transient kinetics:

  • Stopped-flow spectroscopy to identify reaction intermediates

  • Rapid-quench techniques coupled with mass spectrometry for intermediate characterization

• Isotope effects:

  • Deuterium kinetic isotope effects to probe rate-limiting steps

  • Solvent isotope effects to evaluate proton transfer events

• Spectroscopic methods:

  • Circular dichroism to monitor structural changes upon substrate binding

  • Fluorescence spectroscopy with intrinsic tryptophan or extrinsic probes

  • NMR studies for residue-specific interactions with substrate

• Computational approaches:

  • Molecular dynamics simulations of enzyme-substrate interactions

  • Quantum mechanical/molecular mechanical (QM/MM) calculations for transition state energy profiles

These techniques collectively can elucidate the reaction coordinate, identify catalytic residues, and characterize the energetics of urocanate hydration.

How can recombinant hutU be used to study histidine metabolism in O. anthropi?

Recombinant hutU provides valuable tools for investigating histidine metabolism in O. anthropi through several experimental approaches:

• Metabolic flux analysis:

  • Isotope-labeled histidine tracing combined with MS or NMR detection

  • Quantifying metabolic intermediates in wild-type vs. hutU gene knockout strains

• Regulatory studies:

  • Promoter-reporter fusions to study transcriptional regulation of the hut operon

  • Chromatin immunoprecipitation to identify transcription factor binding sites

• Protein-protein interaction studies:

  • Pull-down assays using tagged recombinant hutU to identify interaction partners

  • Bacterial two-hybrid screening for regulatory protein interactions

• In vivo enzyme activity modulation:

  • Controlled overexpression to assess metabolic consequences

  • Competitive inhibitor studies to create chemical knockdowns

These approaches can reveal how O. anthropi regulates histidine utilization in response to environmental conditions and nutrient availability.

What is the relationship between hutU function and antibiotic resistance in O. anthropi?

O. anthropi exhibits intrinsic resistance to multiple antibiotics, particularly β-lactams, as evidenced by high MICs for penicillins, cephalosporins, and aztreonam . While the direct relationship between hutU and antibiotic resistance hasn't been established, several potential connections can be investigated:

• Metabolic adaptation:

  • Histidine metabolism may provide alternative energy sources during antibiotic stress

  • Metabolic byproducts from the hut pathway could potentially modify antibiotics

• Stress response coordination:

  • Gene expression studies to determine if hutU is co-regulated with resistance determinants

  • Analysis of whether antibiotic exposure alters histidine metabolism

• Biofilm formation:

  • Assessment of whether histidine metabolism influences biofilm development, which can enhance antibiotic tolerance

  • Evaluation of hutU knockout effects on biofilm structure and antibiotic susceptibility

Understanding these relationships could provide insights into O. anthropi's ecological adaptations and clinical significance as an emerging opportunistic pathogen .

How can structural comparisons between O. anthropi hutU and human histidine metabolism enzymes inform drug development?

Comparative structural analysis between bacterial hutU and human histidine-metabolizing enzymes offers potential for selective antimicrobial development:

• Key structural differences:

  • Active site architecture comparisons to identify bacterial-specific binding pockets

  • Analysis of cofactor requirements and binding sites unique to bacterial enzymes

  • Evaluation of regulatory domains absent in human counterparts

• Virtual screening approaches:

  • Structure-based docking of compound libraries against O. anthropi hutU

  • Pharmacophore modeling based on substrate and transition state interactions

  • Fragment-based screening for initial scaffold identification

• Selectivity assessment:

  • Counter-screening against human histidine metabolism enzymes

  • Molecular dynamics simulations to evaluate binding specificity

  • Structure-activity relationship studies with promising inhibitor candidates

This approach could potentially identify compounds that selectively target O. anthropi metabolism without affecting human enzymes, providing starting points for novel antimicrobial development against this emerging pathogen with inherent resistance to many conventional antibiotics .

What role might hutU play in the environmental persistence of O. anthropi?

O. anthropi is ubiquitously found in soil, plants, and water sources, including challenging environments like swimming pools and antiseptic solutions . The hutU enzyme may contribute to this remarkable environmental adaptability through:

Research approaches to investigate these aspects include comparative genomics across environmental isolates, transcriptome analysis under various environmental stressors, and in situ metabolic studies using stable isotope probing.

How can system biology approaches integrate hutU function into O. anthropi metabolic networks?

Systems biology offers powerful frameworks for contextualizing hutU within O. anthropi's broader metabolism:

• Genome-scale metabolic modeling:

  • Integration of hutU-catalyzed reactions into constraint-based metabolic models

  • Flux balance analysis to predict growth phenotypes under varying conditions

  • In silico gene knockout studies to assess metabolic robustness

• Multi-omics integration:

  • Correlation of transcriptomics data for hutU with proteomics and metabolomics

  • Network analysis to identify regulatory hubs connected to histidine metabolism

  • Temporal dynamics studies during adaptation to environmental changes

• Comparative systems analysis:

  • Cross-species comparison of histidine utilization network architecture

  • Evolutionary analysis of the hut pathway in relation to ecological niches

  • Identification of species-specific regulatory mechanisms

Such integrative approaches can reveal emergent properties not apparent from studying hutU in isolation and provide insights into O. anthropi's metabolic versatility that contributes to its environmental persistence and opportunistic pathogenicity .

What challenges exist in translating in vitro findings about recombinant hutU to in vivo bacterial physiology?

Translating insights from studies with recombinant partial hutU to understanding its function in living O. anthropi presents several challenges:

• Expression and regulation discrepancies:

  • Native expression levels may differ significantly from recombinant systems

  • Complex transcriptional and post-translational regulation may be absent in vitro

  • Potential for differential subcellular localization affecting function

• Metabolic context considerations:

  • In vivo substrate concentrations may differ from optimal in vitro conditions

  • Presence of competing reactions and metabolic channeling effects

  • Influence of cellular redox state and energy charge on enzyme activity

• Methodological limitations:

  • Challenges in measuring enzyme activity in complex cellular backgrounds

  • Difficulty in distinguishing hutU-specific effects from compensatory responses

  • Technical barriers to creating precise genetic modifications in O. anthropi

• Validation approaches:

  • Development of hutU-specific antibodies for immunolocalization studies

  • Creation of reporter fusions to monitor native expression patterns

  • Implementation of CRISPR-based genome editing for precise genetic manipulation

  • Metabolite profiling with stable isotope labeling to track flux through the pathway

Addressing these challenges requires iterative refinement of both in vitro and in vivo experimental systems, along with computational approaches to bridge the gap between these different levels of biological organization.

How might directed evolution approaches enhance hutU for biotechnological applications?

Directed evolution offers promising strategies for engineering hutU with enhanced properties:

• Potential target properties:

  • Increased catalytic efficiency (higher k_cat/K_m)

  • Enhanced thermostability for industrial applications

  • Modified substrate specificity for novel biotransformations

  • Improved expression and solubility in heterologous hosts

• Directed evolution methodologies:

  • Error-prone PCR to generate mutation libraries

  • DNA shuffling with homologous enzymes from related organisms

  • CRISPR-based systems for in vivo directed evolution

  • Computational design coupled with focused mutagenesis

• Selection/screening strategies:

  • Growth-based selection using histidine auxotrophs

  • High-throughput colorimetric or fluorescent assays

  • Phage display for stability or binding improvements

  • Microfluidic droplet sorting for activity screening

• Potential applications:

  • Biosensors for histidine detection in clinical or food samples

  • Biocatalysis for pharmaceutical intermediate production

  • Environmental remediation of histidine-containing waste

  • Analytical tools for metabolic profiling

The directed evolution approach can systematically address limitations of the wild-type enzyme while exploring novel functionality not present in nature.

What insights could comparative studies between different Ochrobactrum species provide?

Comparative analysis of hutU across Ochrobactrum species can reveal evolutionary adaptations and functional diversity:

• Phylogenetic analysis:

  • Reconstruction of evolutionary relationships between hutU variants

  • Identification of conserved domains versus variable regions

  • Detection of horizontal gene transfer events within the genus

• Structure-function relationships:

  • Correlation of sequence variations with kinetic parameters

  • Identification of species-specific regulatory mechanisms

  • Analysis of co-evolution with other histidine metabolism enzymes

• Ecological correlations:

  • Association of hutU variants with particular ecological niches

  • Connection between enzyme properties and species distribution

  • Potential links to pathogenicity differences between Ochrobactrum species

• Experimental approaches:

  • Heterologous expression of hutU from multiple species

  • Biochemical characterization under standardized conditions

  • Creation of chimeric enzymes to isolate functional domains

  • In vivo complementation studies across species

Such comparative studies could provide fundamental insights into bacterial adaptations while potentially identifying variants with desirable properties for biotechnological applications.

How can emerging technologies advance our understanding of hutU structure and function?

Cutting-edge technologies offer new opportunities to investigate hutU at unprecedented levels of detail:

• Structural biology advancements:

  • Cryo-electron microscopy for high-resolution structures without crystallization

  • Micro-electron diffraction for structural determination from nanocrystals

  • Time-resolved crystallography to capture catalytic intermediates

  • Hydrogen-deuterium exchange mass spectrometry for dynamics studies

• Single-molecule techniques:

  • Förster resonance energy transfer (FRET) to monitor conformational changes

  • Optical tweezers to measure enzyme-substrate interaction forces

  • Single-molecule tracking in living cells to observe in vivo behavior

• Advanced computational methods:

  • Deep learning for improved structure prediction and design

  • Enhanced sampling molecular dynamics for rare event capture

  • Quantum chemistry approaches for transition state modeling

  • Network-based systems biology for contextualizing enzyme function

• Genome editing and synthetic biology:

  • CRISPR-Cas9 for precise genomic modifications in O. anthropi

  • Minimal synthetic pathways incorporating hutU for engineering applications

  • Cell-free expression systems for rapid protein engineering

These technologies, particularly when applied in combination, promise to provide multi-scale understanding from atomic mechanisms to cellular functions and ecological significance of hutU in O. anthropi biology.

What are the current knowledge gaps in understanding O. anthropi hutU?

Despite progress in characterizing O. anthropi as an emerging opportunistic pathogen , significant knowledge gaps remain regarding its hutU enzyme:

• Structural characterization:

  • Three-dimensional structure determination remains incomplete

  • Catalytic mechanism details at the molecular level

  • Potential allosteric regulation sites and mechanisms

• Physiological relevance:

  • Expression patterns under various environmental and clinical conditions

  • Role in biofilm formation and environmental persistence

  • Contribution to virulence or antibiotic resistance phenotypes

• Evolutionary context:

  • Selection pressures shaping hutU sequence in O. anthropi

  • Horizontal gene transfer history within the genus

  • Adaptive significance of sequence variations across strains

• Biotechnological potential:

  • Applicability in industrial biocatalysis

  • Utility as a target for selective inhibitors

  • Potential as a component in biosensors or diagnostic tools

Addressing these gaps requires interdisciplinary approaches combining structural biology, biochemistry, microbial physiology, and computational biology.

What standardized protocols should researchers adopt when working with recombinant hutU?

To ensure reproducibility and facilitate cross-laboratory comparisons, researchers should adopt these standardized protocols:

• Expression and purification:

  • Clearly defined expression constructs with complete sequence information

  • Standardized purification protocols with detailed buffer compositions

  • Consistent quality control metrics (purity, specific activity, homogeneity)

• Activity assays:

  • Defined standard conditions for activity measurements (pH, temperature, buffer)

  • Inclusion of positive controls with known activity

  • Reporting of specific activity alongside protein quantification method

• Storage and handling:

  • Validated storage conditions with stability documentation

  • Freeze-thaw cycle impact assessment

  • Standardized enzyme dilution protocols to minimize variability

• Data reporting:

  • Complete kinetic parameter determination with statistical analysis

  • Detailed methods sections including all buffer components

  • Deposition of sequence and structural data in public databases

Adherence to these standards will accelerate research progress by enabling reliable comparison of results across different studies and research groups.

What integrated research strategies would most effectively advance hutU research?

A comprehensive research strategy for O. anthropi hutU should integrate multiple approaches:

• Multi-disciplinary collaboration:

  • Structural biologists for high-resolution structural determination

  • Enzymologists for detailed kinetic and mechanistic studies

  • Microbiologists for in vivo function and regulation analysis

  • Computational scientists for modeling and systems biology integration

• Technology integration:

  • Combined structural approaches (X-ray, NMR, cryo-EM)

  • Multi-omics profiling under relevant conditions

  • In silico and experimental screening for inhibitors or activators

  • Genetic manipulation with phenotypic characterization

• Translational connections:

  • Clinical isolate collection and comparative genomics

  • Infection model development to study in vivo relevance

  • Exploration of diagnostic or therapeutic applications

  • Environmental sampling to understand ecological distribution

• Open science practices:

  • Data sharing in public repositories

  • Method standardization and protocol sharing

  • Collaborative resource development (antibodies, strains, plasmids)

  • Pre-registration of research questions and approaches