Recombinant Bacillus weihenstephanensis Urocanate hydratase (hutU), partial

What is Bacillus weihenstephanensis and how does it differ from other Bacillus species?

Bacillus weihenstephanensis is a psychrotolerant bacterium belonging to the Bacillus cereus group. Unlike mesophilic B. cereus strains, B. weihenstephanensis can grow at temperatures as low as 7°C and demonstrates poor growth at temperatures above 43°C . The species was first classified in 1998, differentiated from B. cereus by its ability to grow at refrigeration temperatures and by specific signature sequences in the 16S rRNA gene.
Key distinguishing features include:

• Psychrotolerance with demonstrated growth capability at temperatures as low as 7-8°C

• Genomic adaptations with specific signature sequences in 16S rRNA and cspA genes

• Distinct plasmid content, often carrying large plasmids with additional genes for metabolic functions and potentially toxins

• Temperature-dependent sporulation properties, with significantly reduced sporulation efficiency at low temperatures (7-10°C)

• Cold-adapted enzymes with functional activity at lower temperatures
  The fully sequenced strain KBAB4 has become a model organism for studying cold adaptation in Bacilli and contains operons encoding hemolytic (Hbl) and nonhemolytic (Nhe) enterotoxins .

What is the function of urocanate hydratase (hutU) in bacterial metabolism?

Urocanate hydratase (EC 4.2.1.49, also known as imidazolonepropionate hydrolase or urocanate hydratase) catalyzes the second step in the histidine degradation pathway in bacteria . This enzyme specifically converts urocanate to 4,5-dihydro-4-oxo-5-imidazolepropanoate (imidazolonepropionate) through a hydration reaction:
Urocanate + H₂O → Imidazolonepropionate
The reaction is part of the pathway that allows bacteria to utilize histidine as both a carbon and nitrogen source. Urocanate hydratase has several notable biochemical features:

• It binds tightly to NAD+ as a cofactor, which acts as an electrophile in the catalytic mechanism rather than as a redox agent

• The NAD+ is not consumed in the reaction but remains bound to the enzyme

• A conserved cysteine residue is important for catalysis and may be involved in NAD+ binding

• The protein has a molecular weight of approximately 60 kDa
  In B. weihenstephanensis, the hutU gene is typically part of the histidine utilization (hut) operon, which is regulated in response to histidine availability and cellular nitrogen status.

What are the structural characteristics of the urocanate hydratase enzyme?

Urocanate hydratase is a homodimeric protein with each subunit having a molecular weight of approximately 60 kDa . As a member of the carbon-oxygen lyase enzyme family (EC 4.2), specifically hydro-lyases (EC 4.2.1), it catalyzes the elimination of water from its substrate .
Key structural features include:

• Quaternary structure: Functions as a homodimer composed of two identical subunits

• Cofactor binding: Contains tightly bound NAD+ molecules that are critical for catalytic activity but do not undergo redox changes during the reaction

• NAD+-binding domain: Features a Rossmann fold typical of dinucleotide-binding proteins

• Catalytic residues: Includes a conserved cysteine that plays a key role in the reaction mechanism

• Subunit organization: The gene encoding urocanate hydratase (hutU) is found in bacteria and in the liver of many vertebrates, as well as in some plants like Trifolium repens
  In psychrotolerant B. weihenstephanensis, the enzyme likely contains additional structural adaptations that enable activity at lower temperatures compared to mesophilic homologs. These may include more flexible regions, reduced structural stabilization, and an active site configured for catalysis under conditions of lower thermal energy.

How is the hutU gene organized in the B. weihenstephanensis genome?

The hutU gene in B. weihenstephanensis is part of a larger histidine utilization (hut) operon, responsible for histidine catabolism. While specific organization in B. weihenstephanensis is not directly detailed in the available search results, we can draw parallels from related Bacillus species and the information on genetic organization patterns found in this species.
B. weihenstephanensis strain KBAB4 has been fully sequenced, allowing investigation of its genomic organization . The bacterium contains both chromosomal genes and large plasmids, including a 400-kb plasmid containing various genes . This species shows interesting genetic organization patterns, as evidenced by:

• The presence of duplicate genes on plasmids, similar to how Nhe enterotoxin genes exist both on the chromosome and on a 400-kb plasmid

• The association of mobile genetic elements with specialized genes, as seen in the cereulide biosynthesis gene cluster in some B. weihenstephanensis strains that is flanked by IS elements forming a composite transposon (Tnces)

• Expression of genes like sporulation sigma factor sigG and germinant receptor operons (gerI, gerK, gerL, gerR, gerS, and plasmid-located gerS2) under different temperature conditions
  The genomic context of hutU likely influences its expression in response to environmental conditions, particularly temperature, which is a critical factor for this psychrotolerant species. The gene may be subject to both specific regulation related to histidine metabolism and global regulation related to cold adaptation.

What expression systems are optimal for producing recombinant B. weihenstephanensis hutU?

Producing recombinant B. weihenstephanensis hutU requires careful consideration of expression systems that accommodate the psychrotolerant nature of this enzyme. Several expression systems warrant consideration:

• Escherichia coli expression systems:

  • BL21(DE3) with pET vectors provides high-level expression under T7 promoter control

  • Arctic Express strains containing cold-adapted chaperones may facilitate proper folding

  • pCold vectors allow cold-shock induction, which may be particularly suitable for psychrophilic enzymes

  • Expression at lower temperatures (15-20°C) often improves folding and solubility of psychrotolerant proteins

• Bacillus-based expression systems:

  • B. subtilis expression provides a Gram-positive cell environment more similar to the native context

  • The natural sporulation capabilities of Bacillus allow for testing temperature effects similar to those observed in B. weihenstephanensis

  • Secretion-based systems can simplify purification procedures
    Optimization considerations should include:

• Induction temperature: Studies with B. weihenstephanensis have shown significant temperature effects on protein expression and function

• Codon optimization: Adapting the sequence to the preferred codons of the expression host

• Fusion tags: Addition of solubility-enhancing tags (MBP, SUMO) with appropriate cleavage sites

• Temperature history: The temperature at which the expression host is grown affects protein production, as demonstrated by temperature effects on B. weihenstephanensis sporulation
  Testing expression at multiple temperatures is particularly important given that B. weihenstephanensis shows temperature-dependent genomic expression patterns . Evaluating enzyme activity across different expression conditions will help determine optimal parameters for obtaining functional recombinant hutU.

How does temperature affect the catalytic activity of recombinant B. weihenstephanensis hutU?

As an enzyme from a psychrotolerant organism, B. weihenstephanensis hutU likely exhibits distinct temperature-dependent activity profiles compared to mesophilic homologs. Based on temperature effects observed in other B. weihenstephanensis processes, we can anticipate several patterns:

• Temperature optima:

  • Likely lower than mesophilic homologs (possibly 20-25°C vs. 37°C)

  • Broader activity peak across temperature range, enabling function in fluctuating environments

  • May retain significant activity (>70%) even at temperatures as low as 5°C, similar to how B. weihenstephanensis spores can germinate at low temperatures

• Catalytic efficiency (kcat/Km):

  • Higher efficiency at lower temperatures compared to mesophilic homologs

  • Studies with B. weihenstephanensis KBAB4 show temperature-dependent metabolic activities

  • The psychrotolerant nature of B. weihenstephanensis suggests enzymes adapted for function at refrigeration temperatures

• Thermal stability:

  • Lower thermal stability than mesophilic counterparts

  • More rapid inactivation at elevated temperatures (>30°C)

  • Temperature history during preparation likely affects stability, as observed with B. weihenstephanensis spores produced at different temperatures

• Activation energy:

  • Likely lower activation energy for catalysis

  • Compensates for reduced molecular motion at lower temperatures
    Experimental analysis should include measuring enzyme activity across a temperature range (5-40°C) and determining kinetic parameters at each temperature. Research with B. weihenstephanensis KBAB4 demonstrates that temperature affects various cellular processes, including sporulation efficiency, spore heat resistance, and germination capacity . Similar temperature-dependent patterns would be expected for recombinant hutU activity.

What structural adaptations enable B. weihenstephanensis hutU to function at lower temperatures?

As an enzyme from a psychrotolerant organism, B. weihenstephanensis hutU likely possesses structural adaptations that enable it to function efficiently at lower temperatures. These adaptations typically enhance flexibility and catalytic efficiency while potentially sacrificing thermal stability.
Probable structural adaptations include:

• Primary structure modifications:

  • Increased glycine content providing greater backbone flexibility

  • Reduced proline content in loops, increasing local mobility

  • Decreased arginine content, reducing salt bridge formation

  • Potentially increased hydrophobic surface residues, a common feature of cold-adapted proteins

• Active site configurations:

  • More accessible active site to reduce energy barriers for substrate binding

  • Modified substrate binding strength to facilitate product release at low temperatures

  • Preserved positioning of catalytic residues, including the conserved cysteine important for urocanase activity

• Cofactor interactions:

  • Potentially altered NAD+ binding pocket to maintain catalytic efficiency at low temperatures

  • The tight binding of NAD+ (a characteristic of urocanase) may be modulated to balance stability with flexibility
    These adaptations would collectively result in a more flexible enzyme that requires less energy for conformational changes during catalysis, allowing it to function effectively at lower temperatures. B. weihenstephanensis demonstrates temperature-dependent physiological responses , suggesting its enzymes, including hutU, have evolved specific adaptations for function across variable temperature ranges, particularly at the lower end of the spectrum.

What challenges exist in purifying active recombinant B. weihenstephanensis hutU?

Purification of active recombinant B. weihenstephanensis hutU presents several specific challenges that must be addressed to obtain functional enzyme:

• NAD+ retention:

  • Urocanate hydratase requires tightly bound NAD+ for activity, which acts as an electrophile cofactor

  • Harsh purification conditions may cause cofactor loss

  • Supplementation of purification buffers with NAD+ may be necessary to maintain enzyme activity

• Temperature sensitivity:

  • As a psychrotolerant enzyme, stability at room temperature or higher may be limited

  • Purification should be conducted at lower temperatures (4-15°C)

  • B. weihenstephanensis proteins show significant temperature sensitivity, as demonstrated by temperature effects on spore properties

• Expression system considerations:

  • Expression at temperatures higher than the organism's natural growth range may lead to improper folding

  • Improper folding may result in inclusion body formation requiring refolding procedures

  • Temperature history during expression affects protein properties, as seen with B. weihenstephanensis spores

• Activity verification:

  • Spectrophotometric assays for urocanate hydratase activity rely on changes in UV absorption

  • Temperature control during assays is critical for accurate activity determination

  • The psychrotolerant nature of the enzyme requires activity testing at various temperatures

• Stability during storage:

  • Cold-adapted enzymes often show reduced stability during storage

  • Addition of stabilizers (glycerol, sucrose) may be necessary

  • B. weihenstephanensis shows temperature-dependent biological activity patterns that may extend to enzyme stability
    A multi-step purification strategy addressing these challenges is essential for obtaining active enzyme. Lessons from temperature effects on B. weihenstephanensis sporulation, germination, and outgrowth suggest that temperature control throughout the purification process will be crucial for maintaining enzyme activity.

What analytical techniques are most effective for characterizing recombinant B. weihenstephanensis hutU activity?

Several analytical techniques are particularly valuable for characterizing the activity of recombinant B. weihenstephanensis hutU, especially considering its psychrotolerant nature:

• UV-Visible spectroscopy:

  • Direct activity assays: Urocanate absorption decreases at 277 nm during conversion to imidazolonepropionate

  • NAD+ binding studies: Monitoring absorption changes at 340 nm

  • Temperature-controlled cuvette holders are essential for maintaining consistent temperatures during measurement

  • Similar to how B. weihenstephanensis was characterized at different temperatures in growth studies

• Enzyme kinetics analysis:

  • Temperature-dependent kinetic parameters (Km, kcat, kcat/Km)

  • Arrhenius plots to determine activation energy

  • Eyring plots for thermodynamic activation parameters

  • Comparison across multiple temperatures (5-40°C) to identify cold adaptation features

• Thermal stability assessment:

  • Differential scanning calorimetry (DSC) to determine melting temperature

  • Temperature-dependent activity decay measurements

  • Reflects the temperature sensitivity observed in B. weihenstephanensis biological processes

• Structural characterization:

  • Circular dichroism to assess secondary structure stability across temperatures

  • Fluorescence spectroscopy to monitor tertiary structure changes

  • Size-exclusion chromatography to confirm oligomeric state

• Cofactor analysis:

  • Quantification of bound NAD+ per enzyme molecule

  • Fluorescence-based assays for NAD+ binding affinity

  • Critical given the importance of NAD+ as a cofactor for urocanase activity
    Experimental design should incorporate temperature as a key variable, similar to the approach used in studying B. weihenstephanensis KBAB4 sporulation and germination across different temperatures . Careful temperature control during all assays is essential for accurate characterization of this psychrotolerant enzyme.

How can site-directed mutagenesis be used to enhance recombinant B. weihenstephanensis hutU properties?

Site-directed mutagenesis provides a powerful approach to enhance various properties of recombinant B. weihenstephanensis hutU. Strategic modifications can improve stability, activity, or temperature adaptation characteristics:

• Enhancing thermal stability while maintaining low-temperature activity:

  • Introduction of proline residues in loop regions to reduce flexibility

  • Addition of disulfide bridges in non-catalytic regions

  • Introduction of salt bridges at strategic positions

  • These modifications address the typically lower stability of psychrotolerant enzymes like those from B. weihenstephanensis

• Improving catalytic efficiency:

  • Modification of residues involved in substrate binding to optimize Km

  • Altering the microenvironment around the conserved catalytic cysteine residue

  • Engineering the substrate binding pocket for improved substrate recognition

• Optimizing temperature adaptation:

  • Balancing flexibility and stability for broader temperature range activity

  • This approach mirrors the temperature adaptations observed in B. weihenstephanensis, which can function across variable temperatures

  • Targeting regions that may limit activity at lower or higher temperature extremes

• Enhancing NAD+ binding and retention:

  • Modifying residues that interact with the NAD+ cofactor

  • Strengthening the binding of NAD+ to prevent dissociation during purification

  • Critical since NAD+ acts as an essential cofactor for urocanase activity

• Experimental approaches:

  • Individual point mutations based on homology modeling and structural analysis

  • Combinatorial approaches testing multiple mutations simultaneously

  • Validation through kinetic characterization across temperature ranges (5-30°C)
    Site-directed mutagenesis strategies should consider how temperature affects B. weihenstephanensis proteins, as demonstrated by the significant effects of temperature on sporulation, germination, and outgrowth observed in strain KBAB4 . Mutations that enhance function across a broad temperature range would be particularly valuable for applications requiring temperature flexibility.

What methods can detect structural differences between mesophilic and psychrotolerant urocanate hydratases?

Detecting structural differences between mesophilic and psychrotolerant urocanate hydratases requires specialized techniques that can identify subtle adaptations related to temperature sensitivity. The following methods are particularly effective:

• X-ray crystallography:

  • Provides high-resolution structural data to identify key differences

  • Can reveal altered bond distances, solvent-accessible areas, and loop conformations

  • Crystallization may require temperature optimization similar to other procedures with B. weihenstephanensis proteins

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Measures protein dynamics and flexibility differences

  • Can identify regions with altered solvent accessibility

  • Particularly valuable for detecting the increased flexibility characteristic of cold-adapted enzymes

• Molecular dynamics simulations:

  • Models protein motion and flexibility across different temperatures

  • Can predict temperature-dependent structural changes

  • Simulations at different temperatures parallel the experimental approach used with B. weihenstephanensis

• Circular dichroism spectroscopy:

  • Identifies differences in secondary structure content and stability

  • Temperature melting curves reveal stability differences

  • Far-UV and near-UV spectra provide complementary structural information

• Differential scanning calorimetry (DSC):

  • Measures thermal denaturation profiles

  • Quantifies enthalpy of unfolding and melting temperatures

  • Detects domains with different stabilities

• Comparative analysis approach:

  • Side-by-side analysis of B. weihenstephanensis hutU and mesophilic homologs

  • Multiple temperatures for each experiment (5-40°C)

  • Integration of results from multiple techniques for comprehensive comparison
    These techniques would reveal adaptations that enable B. weihenstephanensis hutU to function at lower temperatures, similar to how this species has adapted for psychrotolerant growth . The structural insights gained can guide rational engineering efforts to enhance enzyme properties for specific applications.

How can contradictory kinetic data for recombinant B. weihenstephanensis hutU be reconciled?

Contradictory kinetic data for enzymes like B. weihenstephanensis hutU can arise from various sources, particularly given this organism's psychrotolerant nature and temperature sensitivity. Reconciling such contradictions requires systematic analysis:

• Sources of variability specific to B. weihenstephanensis hutU:

  • Temperature effects: B. weihenstephanensis shows significant temperature-dependent responses in various physiological processes

  • Expression conditions: Temperature history during protein expression affects final properties, as demonstrated with B. weihenstephanensis spores produced at different temperatures

  • NAD+ cofactor content: Variations in bound NAD+ between preparations can cause activity differences

  • Enzyme preparation differences: Purification methods may differentially affect enzyme activity

• Systematic approach to reconcile contradictions:

  • Standardization experiments: Side-by-side testing of different preparations under identical conditions

  • Temperature control validation: Precise temperature monitoring during all assays

  • Cofactor analysis: Quantification of NAD+ content in different enzyme preparations

  • This approach parallels the systematic temperature studies performed with B. weihenstephanensis KBAB4

• Common contradictions and resolutions:

  • Temperature optima discrepancies: Different parameters (Km, kcat) may have different temperature optima

  • Catalytic rate variations: May reflect NAD+ content differences between preparations

  • Stability profile inconsistencies: Different storage conditions affect stability differently

  • Similar to how temperature affects different aspects of B. weihenstephanensis physiology differently

• Data reconciliation methodology:

  • Create a comprehensive experimental conditions database

  • Document all buffer compositions precisely

  • Record temperature control methods

  • Apply statistical approaches to identify significant differences versus experimental noise
    By systematically analyzing the potential sources of variability and employing rigorous experimental approaches with careful temperature control, apparent contradictions in kinetic data can often be reconciled, revealing important insights about this psychrotolerant enzyme's behavior under different conditions.

What statistical approaches are appropriate for analyzing temperature-dependent activity of recombinant B. weihenstephanensis hutU?

Analyzing temperature-dependent enzyme activity requires specialized statistical approaches, particularly for psychrotolerant enzymes like B. weihenstephanensis hutU:

• Arrhenius analysis:

  • Plot ln(k) vs. 1/T (where k is rate constant, T is absolute temperature)

  • Calculate activation energy (Ea) from slope (-Ea/R)

  • Identify non-linear regions indicating temperature-dependent mechanism changes

  • Particularly relevant given B. weihenstephanensis' adaptation to function across temperature ranges

• Eyring-Polanyi analysis:

  • Calculate activation enthalpy (ΔH‡) and entropy (ΔS‡)

  • Determine activation free energy (ΔG‡)

  • Compare thermodynamic parameters with mesophilic homologs

  • Reveals thermodynamic basis of cold adaptation

• Non-linear models for temperature optima:

  • Fit activity data to appropriate non-linear functions

  • Determine temperature optimum (Topt) and operating range

  • Compare with growth temperature range of B. weihenstephanensis (7-43°C)

  • Identify correlation between enzyme and organismal temperature adaptation

• Thermal inactivation kinetics:

  • Calculate half-life at different temperatures

  • Determine inactivation rate constants

  • Compare with thermal stability of B. weihenstephanensis spores formed at different temperatures

  • Model temperature-dependent unfolding equilibria

• Experimental design considerations:

  • Include temperatures spanning B. weihenstephanensis growth range (7-43°C)

  • Fine intervals near expected transition points

  • Multiple replicates at each temperature

  • Parallel analysis of multiple kinetic parameters
    These statistical approaches, when properly applied with appropriate temperature controls, provide rigorous characterization of the temperature-dependent behavior of B. weihenstephanensis hutU and insights into its adaptation mechanisms. The analytical framework should mirror the comprehensive temperature studies performed with B. weihenstephanensis KBAB4, where multiple temperatures were systematically evaluated .

How can recombinant B. weihenstephanensis hutU be utilized in low-temperature biocatalysis applications?

The psychrotolerant nature of B. weihenstephanensis enzymes makes recombinant hutU particularly valuable for low-temperature biocatalysis applications. Several promising applications include:

• Low-temperature biotransformations:

  • Conversion of histidine-related compounds at refrigeration temperatures

  • Reduced energy costs for industrial processes

  • Leverages the demonstrated ability of B. weihenstephanensis to function at temperatures as low as 7°C

  • Particularly valuable for heat-sensitive substrates or products

• Cold-active enzyme cascades:

  • Integration of hutU with other cold-active enzymes for multi-step transformations

  • Creating temperature-optimized pathways for specialized biocatalysis

  • Similar to how B. weihenstephanensis has adapted multiple cellular processes for low-temperature function

• Bioremediation applications:

  • Degradation of histidine-containing contaminants in cold environments

  • Treatment of histidine-rich waste streams at ambient temperatures

  • Winter or cold-climate applications where mesophilic enzymes would be inefficient

• Analytical applications:

  • Biosensors for histidine/urocanate detection functioning at lower temperatures

  • Clinical assays that can be performed at room temperature without heating

  • Extends the temperature range of analytical applications

• Process development considerations:

  • Optimization of reaction conditions specifically for low-temperature catalysis

  • Buffer systems optimized for activity at 5-20°C

  • Integration with other cold-adapted components
    The temperature-dependent properties of B. weihenstephanensis, as demonstrated in comprehensive studies of strain KBAB4 , suggest that its enzymes like hutU would retain significant activity at temperatures where mesophilic homologs would be inefficient, making them valuable tools for low-temperature biocatalytic applications.

What insights does B. weihenstephanensis hutU provide for understanding enzyme cold adaptation mechanisms?

B. weihenstephanensis hutU offers valuable insights into enzyme cold adaptation mechanisms due to the organism's psychrotolerant nature and demonstrated ability to function across a wide temperature range:

• Evolutionary insights:

  • B. weihenstephanensis represents a model for bacterial adaptation to cold environments

  • Comparative analysis with mesophilic Bacillus species reveals evolutionary strategies for cold adaptation

  • The fully sequenced KBAB4 strain enables genomic-level investigation of adaptation mechanisms

• Structure-function relationships:

  • Correlation between structural features and low-temperature activity

  • Identification of flexibility-enhancing modifications that permit catalysis at lower temperatures

  • Comparison with the conserved catalytic mechanism of urocanase across temperature adaptations

• Cofactor interactions in cold adaptation:

  • How NAD+ binding is modulated in cold-adapted enzymes

  • Temperature effects on cofactor-enzyme interactions

  • The role of NAD+ as an electrophilic cofactor in urocanase across temperature ranges

• Activity-stability trade-offs:

  • B. weihenstephanensis exhibits clear temperature-dependent traits, as seen in spore properties

  • Understanding how enzymes balance low-temperature activity with sufficient stability

  • Correlation with organismal growth capabilities across temperature ranges (7-43°C)

• Applications to enzyme engineering:

  • Identifying critical residues and regions for cold adaptation

  • Design principles for engineering cold activity into mesophilic enzymes

  • Understanding how to create enzymes with broad temperature activity ranges

• Comparative systems analysis:

  • Integration with other B. weihenstephanensis cold-adaptation mechanisms

  • Correlation between enzyme properties and cellular physiology

  • Parallels with other temperature-dependent processes in B. weihenstephanensis
    The study of B. weihenstephanensis hutU fits within the broader context of understanding this organism's remarkable adaptation to function at refrigeration temperatures. As shown with studies on KBAB4, B. weihenstephanensis displays comprehensive temperature-dependent physiological responses , making its enzymes valuable models for understanding protein cold adaptation.