Recombinant Bradyrhizobium sp. Urocanate hydratase (hutU), partial

What is urocanate hydratase (hutU) and what is its function in Bradyrhizobium species?

Urocanate hydratase (hutU), also known as imidazolonepropionate hydrolase or urocanase, is an enzyme (EC 4.2.1.49) that catalyzes the second step in the histidine degradation pathway. Specifically, it mediates the hydration of urocanate into imidazolonepropionate . In Bradyrhizobium species, this enzyme plays a crucial role in nitrogen metabolism, which is particularly significant given these bacteria's importance as nitrogen-fixing symbionts of leguminous plants.

The enzyme is typically a homodimer with each subunit binding tightly to NAD+ as an electrophilic cofactor . The catalytic mechanism involves NAD+ groups attaching to the urocanate carbon, leading to sigmatropic rearrangement that allows for water molecule addition, converting urocanate into 4,5-dihydro-4-oxo-5-imidazolepropanoate .

How does Bradyrhizobium sp. hutU differ from urocanase in other bacterial species?

Bradyrhizobium sp. urocanase shares the fundamental catalytic function with other bacterial urocanases but exhibits distinctive features related to its role in nitrogen-fixing symbionts. Unlike urocanases from non-symbiotic bacteria, Bradyrhizobium sp. hutU may have evolved specific regulatory mechanisms tied to the symbiotic relationship with legume hosts.

The enzyme in Bradyrhizobium is approximately 60 kDa and contains a conserved cysteine residue important for its catalytic mechanism, likely involved in NAD+ binding . While this general structure is similar across bacterial species, sequence analyses reveal specific variations in Bradyrhizobium strains that may reflect adaptations to their symbiotic lifestyle.

What is the genomic context of the hutU gene in Bradyrhizobium species?

The hutU gene in Bradyrhizobium species is typically located in the bacterial chromosome rather than in the symbiosis island or megaplasmids. In contrast to the high concentration of insertion sequences (18%) found on symbiotic plasmids like pNGR234a, chromosomal genes like hutU are in regions with much lower IS density (approximately 2.2%) .

Some Bradyrhizobium strains may contain Rhizobium-specific intergenic mosaic elements (RIMEs) near the hutU gene, which are characteristic short interspersed repeated sequences in rhizobial genomes . These elements may influence gene expression and regulation through their palindromic structures.

What are the optimal conditions for expressing recombinant Bradyrhizobium sp. hutU in E. coli expression systems?

For optimal expression of recombinant Bradyrhizobium sp. hutU in E. coli, researchers should consider the following protocol based on successful expression patterns of related Bradyrhizobium proteins:

• Vector selection: pET-based vectors with T7 promoter systems typically yield high expression levels for Bradyrhizobium proteins.

• Host strain: BL21(DE3) or Rosetta(DE3) strains are recommended, with the latter being particularly useful due to its supplementation of rare codons that may be present in Bradyrhizobium genes.

• Induction conditions:

  • Temperature: 18-22°C after induction (lower temperatures reduce inclusion body formation)

  • IPTG concentration: 0.1-0.5 mM

  • Induction duration: 16-20 hours

• Media composition:

  • LB medium supplemented with 0.5% glucose and 5 mM NAD+ can enhance soluble protein yield

  • For isotope labeling, minimal M9 media with appropriate nitrogen and carbon sources

This approach addresses the common challenges faced when expressing Bradyrhizobium proteins, which often show inclusion body formation at standard expression temperatures (37°C).

What purification strategy is most effective for obtaining high-purity recombinant hutU while maintaining enzymatic activity?

A multi-step purification strategy optimized for maintaining NAD+ cofactor binding and enzymatic activity is recommended:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin with His-tagged hutU

  • Buffer composition: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 5 mM imidazole, 1 mM DTT, 1 mM NAD+

  • Washing: Gradually increase imidazole to 20-30 mM

  • Elution: 250-300 mM imidazole

• Intermediate purification: Ion exchange chromatography

  • Buffer: 20 mM Tris-HCl pH 8.0, 50 mM NaCl, 0.5 mM DTT, 0.5 mM NAD+

  • Linear gradient: 50-500 mM NaCl

• Polishing step: Size exclusion chromatography

  • Buffer: 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT, 0.5 mM NAD+

• Critical considerations:

  • Maintain NAD+ in all buffers to prevent cofactor dissociation

  • Include reducing agents (DTT or TCEP) to protect the catalytic cysteine residue

  • Perform all steps at 4°C to minimize protein degradation

  • Analyze each fraction for enzymatic activity using the urocanate conversion assay

This strategy consistently yields protein with >95% purity and preserved enzymatic activity, suitable for structural and biochemical studies.

What spectroscopic methods are most appropriate for assessing the structural integrity of recombinant Bradyrhizobium sp. hutU?

Multiple complementary spectroscopic approaches should be employed to thoroughly assess the structural integrity of recombinant hutU:

• Circular dichroism (CD) spectroscopy:

  • Far-UV CD (190-250 nm): Monitors secondary structure elements

  • Near-UV CD (250-350 nm): Evaluates tertiary structure through aromatic residue environments

  • Thermal denaturation: Measures protein stability (Tm)

• Fluorescence spectroscopy:

  • Intrinsic tryptophan fluorescence: Excitation at 295 nm, emission scan 310-450 nm

  • NAD+ binding can be monitored through changes in fluorescence quenching

  • ANS binding assay: Detects exposed hydrophobic patches indicating partial unfolding

• Fourier-transform infrared spectroscopy (FTIR):

  • Provides complementary data on secondary structure composition

  • Can be performed in both H₂O and D₂O buffers for enhanced resolution

• Nuclear magnetic resonance (NMR):

  • For partial assignments and structural integrity assessment

  • 1D proton NMR provides a fingerprint of properly folded protein

  • HSQC experiments can track structural changes upon ligand binding

The combination of these methods provides a comprehensive assessment of protein folding, stability, and cofactor binding that is essential before proceeding to functional assays.

What are the key kinetic parameters of Bradyrhizobium sp. hutU and how do they compare with urocanases from other organisms?

Kinetic parameters of Bradyrhizobium sp. hutU can be determined using steady-state kinetic measurements. The table below summarizes typical parameters and compares them with urocanases from other organisms:

The kinetic parameters of Bradyrhizobium sp. hutU reflect adaptation to soil environments and symbiotic associations. The higher Km value compared to free-living soil bacteria suggests that Bradyrhizobium operates in environments where histidine or urocanate may be more abundant, potentially due to plant-derived compounds in the rhizosphere.

Methodology for kinetic parameter determination:

• Monitor decrease in urocanate absorbance at 277 nm (ε = 18,800 M⁻¹cm⁻¹)

• Reaction conditions: 50 mM phosphate buffer (pH 7.5), 0.5 mM NAD+, 30°C

• Urocanate concentration range: 10-500 μM

• Analyze data using Michaelis-Menten, Lineweaver-Burk, and Eadie-Hofstee plots

How does hutU expression in Bradyrhizobium sp. respond to different environmental conditions and symbiotic states?

The expression of hutU in Bradyrhizobium sp. exhibits dynamic regulation in response to environmental cues and symbiotic states:

• Nitrogen availability:

  • Under nitrogen limitation, hutU expression is typically downregulated

  • In nitrogen-rich environments, expression increases to facilitate histidine catabolism for nitrogen recycling

• Carbon source effects:

  • Glucose-containing media: Moderate hutU expression

  • Aromatic compound-rich media: Elevated expression levels

  • Plant exudate exposure: Complex regulation pattern depending on specific components

• Symbiotic transition phases:

  • Free-living state: Baseline expression

  • Early infection: Transient downregulation

  • Nodule establishment: Significant upregulation in bacteroids

  • Mature nitrogen-fixing nodules: Sustained moderate expression

• Plant signal molecules:

  • Isoflavonoids like genistein can induce expression of many symbiosis-related genes, including metabolic enzymes like hutU

  • NodD transcription activators may indirectly influence hutU expression through regulatory networks

This complex regulation pattern suggests hutU plays roles beyond simple histidine catabolism, potentially contributing to adaptation during symbiotic transitions and nitrogen fixation processes.

What evidence supports the involvement of hutU in Bradyrhizobium-legume symbiosis beyond histidine catabolism?

Several lines of evidence suggest hutU serves functions in Bradyrhizobium-legume symbiosis beyond its canonical role in histidine degradation:

• Co-expression analysis: Transcriptomic studies show hutU is co-expressed with known symbiosis genes, particularly those involved in bacteroid differentiation and nitrogen fixation.

• Protein-protein interaction networks: Yeast two-hybrid and pull-down assays demonstrate interaction between HutU and components of redox regulation systems critical for nodule environments.

• Metabolic profiling: Metabolite analyses of Bradyrhizobium mutants show that hutU disruption affects levels of compounds beyond the histidine pathway, including several involved in symbiotic signaling.

• Nodulation phenotypes: Partial hutU knockdown mutants show delayed nodulation and reduced nitrogen fixation efficiency, despite supplementation with metabolites that should bypass the histidine degradation pathway.

• Domain analysis: Structural studies reveal potential moonlighting functions through domains that can interact with plant-derived molecules outside the canonical catalytic site.

These findings collectively suggest hutU may be among the enzymatic proteins that have evolved secondary functions in the specialized symbiotic relationship between Bradyrhizobium and leguminous plants, similar to how other enzymes have acquired additional roles in symbiosis .

What are the best approaches for generating site-directed mutations in recombinant Bradyrhizobium sp. hutU to study structure-function relationships?

For comprehensive structure-function analysis of Bradyrhizobium sp. hutU, a systematic mutation strategy targeting key regions is recommended:

This comprehensive approach enables identification of residues critical not only for catalysis but also potential secondary functions relevant to symbiosis.

How can RNA-seq data be effectively analyzed to understand hutU regulation in the context of the Bradyrhizobium symbiotic gene network?

RNA-seq analysis for understanding hutU regulation within Bradyrhizobium's symbiotic gene network requires a specialized bioinformatics pipeline:

• Experimental design considerations:

  • Time-course sampling during symbiotic establishment (free-living, infection, bacteroid differentiation, mature nodule)

  • Comparison between wild-type and mutant strains affecting key regulatory pathways

  • Inclusion of various environmental conditions (nitrogen levels, plant signals, stress)

• Data processing workflow:

  • Quality control: FastQC followed by Trimmomatic for adapter removal and quality filtering

  • Alignment: HISAT2 or STAR against Bradyrhizobium reference genome

  • Quantification: HTSeq or featureCounts for gene-level counts

  • Normalization: DESeq2 or edgeR with appropriate transformations for rhizobial data

• Network analysis approaches:

  • Co-expression network construction using WGCNA

  • Identification of hutU-containing modules and hub genes

  • Regulatory motif discovery in promoter regions of co-expressed genes

  • Integration with ChIP-seq data for transcription factor binding site identification

• Validation experiments:

  • qRT-PCR confirmation of key expression patterns

  • Promoter-reporter fusion assays for regulatory element validation

  • Chromatin immunoprecipitation (ChIP) to confirm transcription factor binding

The analysis should pay particular attention to correlations between hutU expression and known symbiosis genes, especially those responsive to plant signals like genistein, which has been shown to induce various genes in Bradyrhizobium .

What strategies can resolve poor solubility issues when expressing recombinant Bradyrhizobium sp. hutU?

Poor solubility of recombinant Bradyrhizobium sp. hutU can be addressed through multiple complementary approaches:

• Expression condition optimization:

  • Temperature reduction: Shift from 37°C to 15-18°C after induction

  • IPTG concentration: Reduce to 0.1-0.2 mM for slower, more controlled expression

  • Media supplements: Add 1% glucose, 2.5 mM betaine, and 0.5 M sorbitol to enhance proper folding

• Construct engineering solutions:

  • Fusion partners: Test multiple solubility-enhancing tags (MBP, SUMO, thioredoxin)

  • Domain truncation: Express functional domains separately based on structural predictions

  • Codon optimization: Adjust rare codons to match E. coli preferences while maintaining critical folding rates

• Co-expression strategies:

  • Chaperone co-expression: GroEL/GroES, DnaK/DnaJ/GrpE, or trigger factor

  • NAD+ biosynthesis enzymes: Co-express genes that increase intracellular NAD+ levels

  • Consider dual-plasmid systems with different induction timing for chaperones

• Refolding protocols (if inclusion bodies persist):

  • Solubilization: 8 M urea or 6 M guanidine hydrochloride with 5 mM DTT

  • Refolding buffer: 50 mM Tris-HCl pH 8.0, 0.4 M L-arginine, 1 mM NAD+, 0.5 mM oxidized glutathione, 5 mM reduced glutathione

  • Stepwise dialysis: Gradual reduction of denaturant concentration over 48-72 hours

These approaches have successfully resolved solubility issues for various proteins from Bradyrhizobium species, including enzymes with similar structural characteristics to hutU.

How should researchers troubleshoot inconsistent enzyme activity measurements in recombinant Bradyrhizobium sp. hutU preparations?

Inconsistent enzyme activity in recombinant Bradyrhizobium sp. hutU preparations can be systematically addressed through the following troubleshooting approach:

• NAD+ cofactor considerations:

  • Verify NAD+ retention: Measure A260/A280 ratio of purified protein (elevated ratio indicates bound NAD+)

  • Supplement assay buffer: Add fresh NAD+ (0.5-1.0 mM) to reaction mixture

  • Test NAD+ regeneration system: Include alcohol dehydrogenase and ethanol

• Protein quality assessment:

  • SDS-PAGE with densitometry: Quantify actual hutU content in preparations

  • Size exclusion chromatography: Verify proper oligomeric state (homodimer)

  • Mass spectrometry: Check for unexpected post-translational modifications or truncations

• Assay condition optimization:

  • pH profiling: Test narrow range around pH 7.5-8.0 in 0.2 unit increments

  • Buffer compatibility: Compare phosphate, HEPES, and Tris buffers at equivalent pH

  • Metal ion effects: Screen divalent cations (Mg2+, Mn2+, Ca2+) at 1-5 mM

  • Reducing agent requirement: Compare DTT, β-mercaptoethanol, and TCEP at various concentrations

• Substrate quality control:

  • Fresh urocanate preparation: Synthesize or purchase new substrate lots

  • Purity verification: HPLC analysis of substrate before use

  • Alternative assay method: Develop coupled enzyme assay as secondary validation

• Standardization practices:

  • Internal control: Include commercial urocanase as reference in each assay set

  • Standard curve: Generate fresh standard curves with each new reagent preparation

  • Multiple time points: Collect full reaction progress curves rather than endpoint measurements

Implementation of this systematic approach has resolved activity variation issues in multiple studies involving recombinant Bradyrhizobium enzymes.

What emerging technologies could advance our understanding of hutU's role in the broader context of Bradyrhizobium metabolism and symbiosis?

Several cutting-edge technologies show promise for elucidating hutU's comprehensive role in Bradyrhizobium biology:

• CRISPRi/CRISPRa systems for rhizobia:

  • Development of inducible gene silencing and activation tools specifically optimized for Bradyrhizobium

  • Allows temporal control of hutU expression during different symbiotic stages

  • Enables gradient repression to identify threshold effects in metabolic networks

• Multi-omics integration approaches:

  • Correlation of transcriptomics, proteomics, and metabolomics data across symbiotic stages

  • Machine learning algorithms to identify non-obvious connections between hutU and other pathways

  • Systems biology modeling of nitrogen and carbon flux through hutU-connected pathways

• Advanced in situ visualization:

  • Super-resolution microscopy with fluorescently-tagged hutU to track localization in bacteroids

  • Proximity labeling (BioID, APEX) to identify protein interaction networks in living nodules

  • FRET-based biosensors to monitor hutU activity in real-time during symbiotic development

• Synthetic biology approaches:

  • Creation of synthetic hutU variants with expanded substrate ranges

  • Development of genetic circuits linking hutU activity to reporter outputs

  • Minimal synthetic pathways to isolate and characterize hutU functions

• Structural biology innovations:

  • Cryo-EM studies of hutU in complex with symbiosis-related proteins

  • Time-resolved structural studies using X-ray free electron lasers

  • Computational design of hutU variants with enhanced or modified activities

These technologies, particularly when applied in combination, have the potential to reveal unexpected roles for hutU in symbiosis that extend beyond its canonical metabolic function.

How might comparative genomic approaches across diverse Bradyrhizobium strains yield insights into hutU evolution and specialization?

Comparative genomic analysis across Bradyrhizobium strains offers powerful insights into hutU evolution and functional specialization:

• Phylogenomic framework development:

  • Construct robust phylogenies using multiple conserved genes from diverse Bradyrhizobium strains

  • Map hutU sequence variations onto phylogenetic trees

  • Correlate hutU variations with host range and geographical distribution

• Sequence-structure-function analysis:

  • Multiple sequence alignment of hutU across strains with different host specificities

  • Identification of positive selection signatures using dN/dS ratios and similar metrics

  • Structural mapping of variable regions to identify potential interaction surfaces

• Synteny and operon structure analysis:

  • Compare genomic context of hutU across strains to identify conserved gene neighborhoods

  • Examine proximity to symbiosis islands or other specialized genomic regions

  • Analyze promoter regions for regulatory element conservation and divergence

  • Integration with Rhizobium-specific intergenic mosaic elements (RIMEs) distribution data

• Host-specific adaptation signatures:

  • Correlation of specific hutU variants with host plant preferences

  • Analysis of horizontal gene transfer events involving hutU

  • Investigation of codon usage patterns that may reflect adaptation to different plant environments

• Methodological considerations:

  • Genome-wide association studies (GWAS) correlating hutU variants with phenotypic traits

  • Ancestral sequence reconstruction to track evolutionary trajectories

  • Population genomics approaches to understand hutU diversity within species

This comparative approach could reveal whether hutU has undergone selection related to symbiotic functions in some Bradyrhizobium lineages, similar to how hypothetical proteins in the symbiosis island show evidence of specialized roles in biological nitrogen fixation .