Recombinant Rhodopirellula baltica Phosphoribosyl-ATP pyrophosphatase (hisE)

What is Rhodopirellula baltica and why is it significant for enzyme research?

Rhodopirellula baltica SH1(T) is a marine aerobic, heterotrophic bacterium belonging to the phylum Planctomycetes, originally isolated from the Kieler Bight in the southwestern Baltic Sea. Its significance stems from its unique proteome composition and metabolic capabilities that have been extensively characterized through various proteomic analyses. Comprehensive proteome studies have identified 1,267 unique proteins (approximately 17.3% of the total putative protein-coding ORFs), including 261 proteins with predicted signal peptides . This extensive characterization makes R. baltica an excellent model organism for studying specialized prokaryotic enzymes, including those involved in essential metabolic pathways like histidine biosynthesis. When designing experiments with R. baltica enzymes, researchers should consider the organism's native growth conditions and metabolic adaptations to marine environments to optimize expression and characterization protocols.

What is the functional role of Phosphoribosyl-ATP pyrophosphatase (hisE) in bacterial metabolism?

Phosphoribosyl-ATP pyrophosphatase (hisE) catalyzes a critical step in histidine biosynthesis by hydrolyzing phosphoribosyl-ATP to phosphoribosyl-AMP and inorganic pyrophosphate (PPi). This reaction represents an essential regulatory point in the histidine pathway. The enzyme belongs to the broader family of pyrophosphatases, which play crucial roles in cellular metabolism by catalyzing the hydrolysis of pyrophosphate bonds. In prokaryotes like R. baltica, pyrophosphatases can be either soluble (S-PPases) or membrane-bound (M-PPases), with most organisms containing soluble variants that dissipate the energy of pyrophosphate hydrolysis as heat . The study of hisE is particularly valuable for understanding metabolic regulation and energy conservation strategies in different bacterial species. Researchers investigating hisE should examine both its catalytic mechanism and its regulatory context within the larger histidine biosynthesis pathway.

How does R. baltica hisE compare structurally and functionally to homologous enzymes from other bacterial species?

R. baltica hisE shares the core catalytic mechanism with homologous enzymes from other bacterial species, but exhibits unique structural features that reflect its adaptation to marine environments. While specific structural data for R. baltica hisE is limited in the available literature, comparative genomic analyses suggest that prokaryotic pyrophosphatases often exhibit species-specific adaptations while maintaining conserved catalytic domains. Unlike some bacteria that possess membrane-bound pyrophosphatases (such as Rhodospirillum rubrum's H+-PPase), which couple PPi hydrolysis to proton pumping across membranes , the R. baltica hisE is believed to function primarily as a metabolic enzyme without direct coupling to energy conservation. When designing experiments to explore the structure-function relationship of R. baltica hisE, researchers should consider performing comparative enzymatic assays with homologs from both closely related marine bacteria and more distant model organisms like E. coli to identify unique catalytic properties.

What are the optimal E. coli strains for expressing recombinant R. baltica hisE?

The selection of an appropriate E. coli strain is critical for successful expression of R. baltica hisE. Based on comprehensive analysis of recombinant enzyme expression studies, BL21(DE3) and its derivatives are the most widely employed strains, used in approximately 65% of recombinant protein expression cases . These B strains offer several advantages for expressing prokaryotic enzymes like hisE, including deficiency in Lon and OmpT proteases (protecting misfolded proteins from degradation), rapid growth (approximately 20-minute doubling time), and high-level protein synthesis via the T7 expression system .

For potentially difficult-to-express enzymes like hisE, consider the following strain selection strategy:

For R. baltica hisE specifically, researchers should first attempt expression in BL21(DE3), but be prepared to switch to specialized strains if inclusion body formation becomes problematic. Given that only 12% of successful recombinant expression studies utilize K12 derivatives like JM109, DH5α, or MG1655 , these strains should be considered secondary options, primarily when plasmid instability becomes an issue.

What expression vector features best support successful R. baltica hisE production?

The design of expression vectors significantly impacts recombinant R. baltica hisE production. While the search results don't provide specific information about hisE expression vectors, general principles for recombinant enzyme expression can be applied. When selecting or designing an expression vector for R. baltica hisE, consider these key features:

• Promoter strength and inducibility: The T7 promoter system offers strong, controlled expression but may lead to inclusion body formation with fast-folding enzymes like hisE. Consider using tunable promoters like the arabinose-inducible pBAD system for more controlled expression.

• Fusion tags: N-terminal solubility-enhancing tags such as MBP (maltose-binding protein), SUMO, or Thioredoxin can significantly improve the solubility of recombinant enzymes. For purification purposes, smaller affinity tags like His6 or Strep-tag are preferable.

• Codon optimization: Although R. baltica and E. coli are both bacteria, their codon usage patterns differ. Codon harmonization (adjusting codons to match the usage frequency in the host) rather than optimization (using the most abundant codons) may better preserve proper folding kinetics .

• Vector copy number: Lower copy number plasmids often result in more controlled expression, reducing the risk of inclusion body formation that can occur with high-copy vectors like those with pUC origins.

The methodology for testing multiple vector configurations should involve a systematic approach, starting with a small-scale expression screening using various combinations of these features to identify optimal conditions before scaling up.

How can inclusion body formation be minimized when expressing R. baltica hisE in E. coli?

Inclusion body formation is a common challenge when expressing recombinant enzymes in E. coli. For R. baltica hisE, a multi-faceted approach can minimize this issue:

• Temperature modulation: Lowering the expression temperature to 16-25°C significantly slows protein synthesis, allowing more time for proper folding. Implementation should include pre-cooling cultures before induction and maintaining precise temperature control throughout expression.

• Induction optimization: Reducing inducer concentration (e.g., using 0.1 mM IPTG instead of 1 mM) and extending expression time can promote proper folding. A methodical approach involves testing an induction matrix:

• Co-expression with chaperones: Expression vectors that co-express molecular chaperones (GroEL/GroES, DnaK/DnaJ/GrpE) can significantly enhance proper folding. When implementing this approach, consider using compatible plasmids with different antibiotic selection markers and origins of replication.

• Culture media engineering: Supplementing growth media with osmolytes (glycerol, sorbitol) or specific metal ions required for enzyme function can improve folding. For R. baltica enzymes adapted to marine environments, adding 1-3% NaCl to the growth medium may better mimic native conditions and improve folding .

These approaches should be tested systematically, starting with small-scale cultures to identify optimal conditions before scaling up production. Successfully expressed soluble protein should be verified by both SDS-PAGE and activity assays to confirm proper folding and function.

How can systems biology approaches enhance our understanding of R. baltica hisE function in heterologous hosts?

Systems biology approaches offer powerful tools for understanding the broader impacts of expressing R. baltica hisE in heterologous hosts like E. coli. When R. rubrum H+-PPase was expressed in E. coli as the sole pyrophosphatase, 13C-metabolic flux analysis (13C-MFA) revealed a significant 36% decrease in tricarboxylic acid (TCA) cycle fluxes compared to wild-type E. coli MG1655 . This finding demonstrates how heterologous pyrophosphatase expression can dramatically alter cellular metabolism.

For R. baltica hisE research, implementing similar systems approaches involves:

• Metabolic flux analysis: Apply 13C-MFA to track carbon flow through central metabolism when expressing hisE, focusing on:

  • Changes in histidine pathway flux

  • Alterations in energy metabolism (ATP/ADP ratios)

  • Shifts in related amino acid biosynthesis pathways

• Transcriptomics integration: RNA-seq analysis before and after hisE induction can reveal compensatory mechanisms and stress responses in the host. Key data points to collect include:

  • Changes in expression of E. coli's native pyrophosphatases

  • Alterations in histidine biosynthesis gene expression

  • Global stress responses triggered by heterologous expression

• Proteomics: Compare the host proteome with and without hisE expression, similar to the comprehensive proteome analysis performed for R. baltica itself, which identified 1,115 nonredundant proteins . This approach can identify unexpected protein-protein interactions and regulatory effects.

The methodological implementation should involve careful experimental design with biological replicates, appropriate controls, and integrated data analysis pipelines that can correlate changes across multiple 'omics datasets.

What are the kinetic differences between native and recombinant R. baltica hisE, and how can these differences be characterized?

Characterizing kinetic differences between native and recombinant R. baltica hisE requires sophisticated enzymological approaches. While specific data for R. baltica hisE is not provided in the search results, studies of other recombinant enzymes indicate that expression conditions can significantly alter kinetic parameters.

A comprehensive kinetic characterization methodology should include:

• Steady-state kinetics: Determine key parameters including:

  • Km and kcat for phosphoribosyl-ATP

  • Inhibition constants for products and pathway intermediates

  • pH-activity profile and optimal conditions

• Pre-steady-state kinetics: Use stopped-flow spectroscopy to identify rate-limiting steps in the catalytic mechanism, particularly:

  • Substrate binding rates

  • Conformational changes

  • Product release kinetics

• Comparative analysis: Express R. baltica hisE in multiple systems (E. coli B and K12 strains, cell-free systems) and compare:

• Structure-function correlation: Combine kinetic data with structural analysis (X-ray crystallography or cryo-EM) to identify how expression system-dependent differences in post-translational modifications or folding affect catalytic site geometry.

The execution of these experiments requires careful enzyme preparation with consistent purification protocols across all samples to ensure valid comparisons.

How does the expression of R. baltica hisE in E. coli impact the host cell's energy metabolism?

The expression of pyrophosphatase enzymes can significantly impact host cell energy metabolism. When H+-PPase from R. rubrum was expressed in E. coli as the sole pyrophosphatase, it led to a 36% decrease in TCA cycle fluxes compared to wild-type E. coli . This substantial metabolic shift suggests that heterologous pyrophosphatases can redirect energy flows within the host cell.

For R. baltica hisE, researchers should investigate:

• Energy parameters: Measure key bioenergetic indicators before and after induction:

  • ATP/ADP ratios using luciferase-based assays

  • NAD+/NADH and NADP+/NADPH ratios

  • Membrane potential using fluorescent probes

• Carbon flux distribution: Implement 13C-MFA to quantify:

  • Changes in glycolytic flux

  • TCA cycle activity

  • Pentose phosphate pathway utilization

  • Anabolic pathway activities

• Growth characteristics: Monitor detailed growth parameters in various media compositions:

  • Lag phase duration

  • Exponential growth rate

  • Final biomass yield

  • Metabolite secretion profiles

A properly designed experimental approach should include isogenic control strains expressing catalytically inactive hisE variants to distinguish between effects caused by enzyme activity versus protein expression burden.

What analytical techniques are most effective for confirming the structural integrity of recombinant R. baltica hisE?

Confirming the structural integrity of recombinant R. baltica hisE requires a multi-technique approach that examines the enzyme at different levels of structural organization:

• Primary structure verification:

  • Mass spectrometry (MS): Perform intact protein MS to confirm the exact molecular weight

  • Peptide mapping: Use tryptic digestion followed by LC-MS/MS to verify sequence coverage

  • N-terminal sequencing: Confirm the start of the protein, particularly important if signal peptide processing is suspected

• Secondary and tertiary structure analysis:

  • Circular dichroism (CD): Quantify secondary structure elements (α-helices, β-sheets)

  • Fluorescence spectroscopy: Measure intrinsic tryptophan fluorescence to assess tertiary folding

  • Differential scanning calorimetry: Determine thermal stability and folding cooperativity

• Quaternary structure and aggregation assessment:

  • Size exclusion chromatography with multi-angle light scattering (SEC-MALS): Determine oligomeric state and homogeneity

  • Dynamic light scattering: Detect early aggregation and polydispersity

  • Analytical ultracentrifugation: Provide definitive data on oligomeric state and shape

• Functional integrity:

  • Enzyme activity assays: Compare specific activity to theoretical maximum

  • Ligand binding studies: Use isothermal titration calorimetry or surface plasmon resonance

  • Inhibitor sensitivity profiling: Compare inhibition patterns with known standards

The implementation methodology should follow a hierarchical approach, starting with basic integrity checks (SDS-PAGE, Western blot) before progressing to more sophisticated structural analyses. All data should be compared against in silico predictions based on homologous enzymes.

How can isotopic labeling enhance structural studies of recombinant R. baltica hisE?

Isotopic labeling provides powerful tools for detailed structural and functional studies of recombinant R. baltica hisE:

• NMR spectroscopy applications:

  • Uniform 15N and 13C labeling: Express hisE in minimal media with 15NH4Cl and 13C-glucose as sole nitrogen and carbon sources to enable multidimensional NMR experiments for complete structure determination

  • Selective amino acid labeling: Label specific residues in the active site to monitor catalytic mechanisms

  • TROSY experiments: For larger enzyme complexes, employ transverse relaxation-optimized spectroscopy with 2H/15N/13C triple labeling

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

  • Methodology: Express unlabeled protein, then expose to D2O buffer for various time periods

  • Data analysis: Regions with rapid hydrogen exchange indicate exposed/flexible regions

  • Application: Map substrate binding sites and conformational changes during catalysis

• Neutron crystallography:

  • Sample preparation: Express hisE in fully deuterated media

  • Advantage: Visualize hydrogen atoms in the catalytic mechanism

  • Implementation: Requires specialized facilities but provides unique mechanistic insights

The execution of these experiments requires careful optimization of expression protocols to ensure high incorporation rates of isotopic labels without compromising protein folding or yield. For NMR studies particularly, E. coli strains optimized for isotopic labeling (like BL21(DE3) auxotrophs) should be considered.

What high-throughput methods can accelerate optimization of R. baltica hisE expression conditions?

High-throughput methods can dramatically accelerate the optimization of expression conditions for R. baltica hisE:

• Parallel expression screening:

  • Implement a factorial design combining key variables:

    • Expression strain (BL21(DE3), Rosetta, Arctic Express)

    • Induction temperature (16°C, 25°C, 30°C, 37°C)

    • Inducer concentration (0.01-1.0 mM IPTG range)

    • Media composition (LB, TB, auto-induction, minimal)

  • Use 96-well deep-well plates with breathable seals for parallel cultures

  • Analyze expression via automated cell lysis and SDS-PAGE or dot-blot analysis

• Automated solubility assessment:

  • Split-GFP complementation system: Fuse hisE to a split-GFP fragment; fluorescence indicates soluble expression

  • In-cell fluorescence monitoring: Use plate readers for real-time tracking of expression

  • Automated fractionation: Separate soluble and insoluble fractions with robotic liquid handlers

• Activity-based screening:

  • Develop a colorimetric or fluorescent assay for hisE activity

  • Implement in a 384-well format for rapid condition screening

  • Couple with automated image analysis for quantification

• Design of Experiments (DoE) approach:

  • Rather than testing all possible combinations, use statistical DoE

  • Start with screening designs to identify significant factors

  • Follow with response surface methodology to optimize within narrower ranges

Implementation should include automated data collection and analysis pipelines to rapidly identify optimal conditions from thousands of potential combinations.

How can site-directed mutagenesis be used to enhance the catalytic efficiency of R. baltica hisE?

Site-directed mutagenesis represents a powerful approach for enhancing the catalytic properties of R. baltica hisE. While specific mutagenesis data for this enzyme is not available in the search results, a methodical approach based on enzyme engineering principles can be outlined:

The experimental implementation should include careful controls and standardized conditions to ensure valid comparisons between enzyme variants. Results should be analyzed in the context of structural models to develop iterative improvements.

What computational approaches can predict optimal conditions for R. baltica hisE solubility and activity?

Computational approaches offer valuable tools for predicting optimal conditions for R. baltica hisE expression, solubility, and activity:

• Sequence-based predictions:

  • Solubility prediction algorithms: Use tools like Protein-Sol, ccSol, and PROSSO to estimate intrinsic solubility

  • Aggregation propensity: Employ AGGRESCAN or TANGO to identify aggregation-prone regions

  • Disorder prediction: Apply IUPred or PONDR to identify flexible regions that may impact folding

• Structural bioinformatics:

  • Homology modeling: Generate 3D models using related crystal structures as templates

  • Molecular dynamics simulations: Assess stability under different solvent conditions

  • Normal mode analysis: Identify conformational flexibility that may impact folding

• Expression optimization tools:

  • Codon usage analysis: Use tools like OPTIMIZER or ATGme for codon harmonization

  • mRNA secondary structure prediction: Assess 5' UTR and early coding sequence for translation efficiency

  • Signal sequence prediction: Identify potential processing sites that could affect expression

• Machine learning approaches:

  • Train models on existing protein expression datasets

  • Integrate multiple features (charge distribution, hydrophobicity, etc.)

  • Validate predictions with small-scale expression tests

The implementation methodology should combine these computational approaches with experimental validation in an iterative process. Initial predictions should guide experimental design, with results feeding back to refine computational models.

How can cryo-electron microscopy be applied to study the structure-function relationship of R. baltica hisE?

Cryo-electron microscopy (cryo-EM) offers powerful capabilities for studying the structure-function relationship of R. baltica hisE, particularly for capturing different conformational states during catalysis:

• Sample preparation strategy:

  • Protein purity: Achieve >95% homogeneity through rigorous purification

  • Concentration optimization: Typically 1-3 mg/mL for single-particle analysis

  • Vitrification conditions: Test multiple grids (Quantifoil, C-flat) and blotting parameters

  • Substrate/inhibitor complexes: Prepare samples with various ligands to capture different states

• Data collection methodology:

  • Microscope selection: Use high-end instruments (Titan Krios, Glacios) with direct electron detectors

  • Collection parameters: Optimize defocus range, exposure, and framing strategy

  • Automation: Implement SerialEM or EPU for efficient data acquisition

  • Processing pipeline: Use RELION, cryoSPARC, or EMAN2 for image processing

• Conformational dynamics studies:

  • Time-resolved cryo-EM: Mix enzyme and substrate using microfluidic devices before vitrification

  • Classification strategies: Implement 3D classification to separate different conformational states

  • Energy landscape mapping: Combine multiple states to visualize the catalytic cycle

• Integration with other methods:

  • Molecular dynamics: Use cryo-EM structures as starting points for simulation

  • Mutagenesis validation: Test predictions from structures with targeted mutations

  • Small-angle X-ray scattering: Validate solution-state conformations

This approach requires significant expertise and access to advanced instrumentation, but can provide unprecedented insights into the catalytic mechanism of R. baltica hisE by visualizing conformational changes during substrate binding and catalysis.

What are the most promising future research directions for understanding and utilizing R. baltica hisE?

The study of recombinant Rhodopirellula baltica Phosphoribosyl-ATP pyrophosphatase (hisE) offers several promising research directions that could significantly advance our understanding of enzyme function and application:

These research directions represent opportunities for both fundamental discoveries about enzyme function and practical applications in biotechnology and synthetic biology.

How does understanding R. baltica hisE contribute to broader knowledge of metabolic pathway evolution?

The study of R. baltica hisE provides unique insights into metabolic pathway evolution, particularly regarding essential biosynthetic pathways in diverse bacterial lineages:

• Evolutionary adaptation: As a member of the Planctomycetes phylum, R. baltica represents a distinct evolutionary lineage with unique adaptations to marine environments. Studying hisE in this context can reveal how essential metabolic enzymes adapt to specific ecological niches while maintaining core catalytic functions. Comparative analysis of hisE sequences and structures across bacterial phyla can illuminate how selection pressures have shaped enzyme evolution.

• Metabolic pathway integration: Histidine biosynthesis intersects with nucleotide metabolism, highlighting how metabolic pathways coevolve as integrated networks rather than isolated units. The dual role of pyrophosphatases in both specific biosynthetic pathways and broader energy metabolism exemplifies the interconnected nature of cellular systems. The significant metabolic shifts observed when heterologous pyrophosphatases are expressed in E. coli, such as the 36% decrease in TCA cycle flux seen with R. rubrum H+-PPase , demonstrates how seemingly isolated enzymatic changes can have broad metabolic consequences.

• Methodological advances: The comprehensive proteomic approaches that identified 1,267 unique proteins in R. baltica provide a model for how integrated 'omics approaches can reveal evolutionary patterns in metabolic networks. These methodologies allow researchers to place individual enzymes like hisE within their broader metabolic context, revealing co-evolutionary patterns and functional constraints.

By studying R. baltica hisE within this evolutionary framework, researchers can better understand how essential metabolic functions are maintained across diverse bacterial lineages while adapting to specific environmental conditions.

What standardized protocols should be established to improve reproducibility in R. baltica hisE research?

To address the lack of standardized methods noted in recombinant enzyme expression research , the following protocols should be established for R. baltica hisE studies:

• Expression system standardization:

  • Define a primary reference strain (BL21(DE3)) and vector system

  • Establish standard growth conditions and media compositions

  • Develop a tiered approach for troubleshooting expression issues

  • Create reference standards for protein yield and activity benchmarks

• Purification protocol standardization:

  • Define a preferred affinity tag system with validated cleavage methods

  • Establish quality control metrics (purity, activity, oligomeric state)

  • Develop storage and stability protocols for consistent enzyme preparations

  • Create standard buffer systems optimized for activity and stability

• Activity assay standardization:

  • Establish primary and secondary assay methods with defined units

  • Define standard reaction conditions (temperature, pH, ionic strength)

  • Develop calibration standards for inter-laboratory comparison

  • Create detailed protocols for kinetic parameter determination

• Data reporting requirements:

  • Comprehensive expression conditions reporting template

  • Standardized format for kinetic parameter reporting

  • Minimum information requirements for structural studies

  • Metadata standards for computational models and predictions