Recombinant Photobacterium profundum 6,7-dimethyl-8-ribityllumazine synthase (ribH)

What is the optimal expression system for recombinant Photobacterium profundum ribH?

The optimal expression system for recombinant Photobacterium profundum ribH depends on your experimental goals. For structural studies requiring high yields of soluble protein, E. coli BL21(DE3) with pET-based vectors typically provides good expression levels when induced at lower temperatures (16-18°C). For functional studies where post-translational modifications may be important, consider using the native Photobacterium profundum expression system with vectors like pFL122 that have been successfully used for expressing Photobacterium profundum genes . When designing your expression construct, include a gel purification step of the vector containing your gene of interest, similar to how P. profundum 3TCK sequencing library containing various genes was processed with restriction enzymes followed by gel purification before ligation into the appropriate vector .

What are the key buffer conditions for maintaining ribH stability during purification?

For maintaining ribH stability during purification, use buffers containing 50 mM Tris-HCl (pH 7.5-8.0), 150-300 mM NaCl, 10% glycerol, and 5 mM β-mercaptoethanol or 1 mM DTT to prevent oxidation of cysteine residues. As Photobacterium profundum is a deep-sea bacterium adapted to high pressure environments, consider adding osmolytes like trimethylamine N-oxide (TMAO) at 1-2% to mimic its native high-pressure environment and enhance protein stability. During purification, maintain temperatures between 4-8°C and add protease inhibitors (e.g., PMSF at 1 mM) to prevent degradation. For long-term storage, flash freeze aliquots in liquid nitrogen and store at -80°C with additional glycerol (up to 20%).

How can I assess the catalytic activity of purified ribH enzyme?

The catalytic activity of purified ribH enzyme can be assessed using a spectrophotometric assay that monitors the formation of 6,7-dimethyl-8-ribityllumazine from 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy-2-butanone 4-phosphate. The reaction can be followed by measuring the increase in absorbance at 408-410 nm, which corresponds to the formation of the product. A standard reaction mixture typically contains:

• 100 mM potassium phosphate buffer (pH 7.0)

• 5 mM MgCl₂

• 100 μM 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione

• 100 μM 3,4-dihydroxy-2-butanone 4-phosphate

• 0.1-1 μg purified ribH enzyme

Incubate at 25°C and record absorbance readings at 1-minute intervals for 10-15 minutes. Calculate the enzyme activity in terms of μmol product formed per minute per mg of enzyme (specific activity). This method allows you to compare the catalytic efficiency of wild-type and mutant forms of the enzyme, similar to how catalytic efficiencies were compared in evolved enzyme variants in other studies (kcat/KM values) .

What considerations should be made when designing site-directed mutagenesis experiments for ribH?

When designing site-directed mutagenesis experiments for ribH, consider the following methodological approaches:

• Target selection: Focus on residues in the active site that directly interact with substrates, as well as second-shell residues that may influence active site geometry and dynamics. Based on approaches used in other enzyme studies, both first-shell (directly contacting substrate) and second-shell residues can significantly impact catalytic efficiency .

• Conservation analysis: Compare ribH sequences across different bacterial species to identify highly conserved residues, which likely play crucial roles in enzyme function or structural integrity.

• Structural considerations: If crystal structures are available, use them to identify residues involved in substrate binding, catalysis, or maintaining the active site architecture. Consider the potential impact of mutations on protein stability and folding.

• Mutation types: Design conservative mutations (e.g., Asp to Glu) to probe subtle effects on catalysis, and more radical changes to test mechanistic hypotheses. Consider examining both single and combinatorial mutations, as synergistic effects may emerge from multiple substitutions .

• Control experiments: Always include appropriate positive (wild-type) and negative (catalytically inactive) controls in your assays to ensure reliable interpretation of results.

• Conformational effects: Consider how mutations might alter the conformational ensemble of the enzyme, as changes in protein dynamics can significantly impact catalytic efficiency even when mutations are distal from the active site .

How does the conformational ensemble of ribH impact its catalytic efficiency, and what techniques can be used to characterize these dynamics?

The conformational ensemble of ribH significantly impacts its catalytic efficiency by influencing substrate binding, transition state stabilization, and product release. Recent research on enzyme design has demonstrated that catalytic residues become increasingly rigidified through improved packing during evolutionary optimization, leading to better pre-organization of the active site to favor productive substrate binding . To characterize these dynamics:

• Room-temperature X-ray crystallography: This technique reveals the conformational ensemble that the enzyme samples at physiologically relevant temperatures. Unlike cryo-cooling methods, room-temperature crystallography captures the distribution of conformational states, providing insight into protein flexibility relevant to catalysis . For ribH, this approach could identify mobile loops or residues that undergo conformational changes during catalysis.

• NMR spectroscopy: 15N-HSQC and CPMG relaxation dispersion experiments can map conformational exchange processes occurring on microsecond-to-millisecond timescales, which often correlate with catalytic steps.

• Molecular dynamics (MD) simulations: All-atom MD simulations can model the conformational landscape of ribH, particularly when validated against experimental data. These simulations can reveal how mutations alter the energy landscape and population of catalytically competent sub-states.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): This technique provides information about protein dynamics by measuring the rate of hydrogen exchange with deuterium in different regions of the protein.

The impact of conformational dynamics on catalysis could be evaluated by comparing wild-type ribH with rationally designed mutants targeting regions involved in conformational changes. As observed in other enzyme systems, directed evolution often yields beneficial mutations at positions remote from the active site, which enhance catalysis by shifting conformational populations toward catalytically active sub-states .

What approaches can be used to engineer increased thermostability in Photobacterium profundum ribH while maintaining catalytic efficiency?

Engineering increased thermostability in Photobacterium profundum ribH while maintaining catalytic efficiency requires balancing rigidity for stability with the flexibility needed for catalysis. Several approaches can be implemented:

• Consensus-based design: Align multiple ribH sequences from thermophilic organisms and identify consensus amino acids at variable positions. Introducing these consensus residues into P. profundum ribH may increase thermostability without compromising function.

• Computational design using ensemble modeling: Instead of using a single template structure for computational design, employ an ensemble of backbone templates derived from experimental data, as this approach has shown success in capturing the conformational flexibility necessary for optimal enzyme function . When introducing stabilizing mutations, evaluate their impact across the entire conformational ensemble rather than just on a single structure.

• Introduction of salt bridges and disulfide bonds: Strategically place new ionic interactions or disulfide bonds to stabilize flexible regions without restricting catalytically essential movements. Focus particularly on surface-exposed loops that contribute to instability.

• Active site rigidification: As observed during the evolution of other enzymes, catalytic residues become increasingly rigidified through improved packing, leading to better pre-organization of the active site . Engineer second-shell mutations that improve packing around catalytic residues while maintaining their optimal geometry.

• Iterative approach to optimization: Rather than attempting to introduce all stabilizing mutations simultaneously, use an iterative approach where each round of mutations is followed by experimental validation. This allows for the detection and correction of unexpected negative interactions between mutations.

Evaluation should include differential scanning calorimetry (DSC) to determine melting temperatures, long-term activity assays at elevated temperatures, and structural analysis to confirm that the engineered variants maintain the correct fold and active site geometry.

How can contradictions in catalytic efficiency data between different experimental setups for ribH be resolved?

Resolving contradictions in catalytic efficiency data between different experimental setups for ribH requires a systematic approach to identify and address variables that might contribute to discrepancies. Here's a methodological framework:

• Standardize reaction conditions: Ensure that all comparative experiments use identical buffer systems, pH, temperature, substrate concentrations, and enzyme preparation methods. Small variations in these parameters can significantly affect kinetic measurements.

• Employ multiple analytical methods: Validate results using orthogonal techniques. For example, if discrepancies exist in spectrophotometric assays, complement these with HPLC-based product quantification or isothermal titration calorimetry (ITC) for binding studies.

• Analyze enzyme conformational states: As demonstrated in research on enzyme design, proteins sample multiple conformational sub-states, and the population distribution of these states can significantly impact catalytic activity . Use room-temperature X-ray crystallography to capture the conformational ensemble under different experimental conditions, potentially revealing why certain setups yield different results.

• Develop a robust explanation model: When contradictions are detected, develop a framework similar to the Red Teaming approach used for contradiction analysis, where you systematically identify the contradictory elements, provide explicit explanations for the contradiction, and suggest modifications to reconcile the conflicting data . This structured approach ensures thorough analysis of all potential sources of discrepancy.

• Control for experimental artifacts: Check for common issues that can affect enzyme assays:

  • Enzyme oligomerization state (use size-exclusion chromatography to verify)

  • Presence of inhibitory compounds in buffers or substrates

  • Time-dependent enzyme inactivation

  • Product inhibition effects

• Statistical validation: Implement rigorous statistical analysis of replicate experiments, including outlier detection and appropriate significance testing to determine whether apparent contradictions are statistically meaningful.

By methodically addressing these aspects, researchers can identify whether contradictions stem from experimental variations, reflect genuine biological phenomena, or result from technical artifacts.

What is the impact of pressure on the structure and function of Photobacterium profundum ribH, and how can high-pressure enzymatic assays be designed?

The impact of pressure on Photobacterium profundum ribH structure and function is particularly relevant given that P. profundum is a piezophilic (pressure-loving) deep-sea bacterium. Pressure can affect enzyme kinetics, protein folding, and quaternary structure. To investigate these effects and design high-pressure enzymatic assays:

• High-pressure spectroscopic analysis: Utilize high-pressure optical cells coupled with UV-Vis spectrophotometry to monitor ribH activity under varying pressures. The reaction can be monitored at 408-410 nm to track 6,7-dimethyl-8-ribityllumazine formation under pressures ranging from atmospheric to 1000 bar (equivalent to deep-sea environments).

• Structural adaptations assessment: Compare the amino acid composition and structural features of P. profundum ribH with homologous enzymes from non-piezophilic organisms to identify potential pressure-adaptive features such as:

  • Increased glycine content in loop regions

  • Reduced hydrophobic core volume

  • Enhanced ion pair networks

  • Modified surface charge distribution

• Experimental design considerations for high-pressure assays:

  • Use pressure-resistant materials for reaction vessels

  • Employ buffer systems with minimal pressure-dependent pH shifts

  • Account for pressure effects on substrate and cofactor solubility

  • Consider temperature-pressure compensation (pressure increase often requires temperature adjustment to maintain equivalent reaction conditions)

• Pressure-jump experiments: Design experiments that rapidly change pressure during catalysis to detect pressure-dependent rate-limiting steps in the reaction mechanism.

• In vivo relevance: Link in vitro high-pressure studies to in vivo function by comparing with P. profundum growth and metabolic characteristics under pressure, similar to how UV irradiation effects have been tested on P. profundum strains under various recovery conditions .

A comprehensive understanding of pressure effects would require correlation with conformational dynamics studies, as pressure can shift the conformational ensemble of enzymes toward more compact states, potentially affecting catalytic efficiency by altering the population of catalytically competent sub-states .

Protocol for Photoreactivation Studies with Photobacterium profundum Expressing Recombinant ribH

When studying the potential role of ribH in DNA repair through photoreactivation mechanisms in Photobacterium profundum, researchers can adapt established protocols for photoreactivation studies. The following methodology is based on successful approaches with P. profundum:

• Culture preparation:

  • Grow late exponential cultures of P. profundum strains (wild-type and ribH-modified strains) in 75% strength 2216 Marine Broth

  • Prepare triplicate dilution series for each strain condition

• Experimental conditions:

  • Control group: Untreated cultures

  • UV treatment group: Cultures irradiated without blue light recovery

  • Photoreactivation group: UV-irradiated cultures with blue light recovery

• UV irradiation procedure:

  • Expose cell cultures uncovered to a germicidal lamp with emission peak at 253.7 nm (such as Philips G25 T8)

  • Use an irradiation duration of 10 seconds at a power of 220 μW/cm²

  • Measure irradiance levels using a calibrated digital radiometer (e.g., Spectroline DM-365 XA)

• Photoreactivation conditions:

  • Following UV exposure, cover the plates to filter shorter wavelength radiation

  • Allow cells to recover for 1 hour under "black" light (such as Philips TLD 15 W/08) emitting in the 350-400 nm range

  • Maintain irradiance at 20 μW/cm²

• Survival assessment:

  • Count colony-forming units (CFUs) for each condition

  • Calculate survival rates relative to unirradiated controls

  • Analyze data for statistical significance using ANOVA with post-hoc tests

• Gene expression analysis:

  • Extract RNA from cells at different time points during recovery

  • Perform RT-qPCR to quantify ribH expression changes in response to UV damage

  • Compare with expression of known photolyase genes like phr

This experimental design allows for the assessment of whether modifications to ribH expression affect DNA repair capabilities and survival following UV damage, potentially revealing connections between riboflavin synthesis and DNA repair mechanisms in P. profundum.

Method for Analyzing Conformational Ensembles of ribH Using Room-Temperature X-ray Crystallography

Room-temperature X-ray crystallography provides valuable insights into the conformational ensemble of enzymes under physiologically relevant conditions. The following protocol outlines how to apply this method to ribH from Photobacterium profundum:

• Crystal preparation:

  • Grow high-quality crystals of purified ribH using hanging drop or sitting drop vapor diffusion

  • Optimize crystallization conditions to obtain crystals suitable for room-temperature data collection

  • For comparative studies, prepare crystals of wild-type ribH and engineered variants

• Room-temperature mounting:

  • Mount crystals in thin-walled capillaries or specialized room-temperature sample holders

  • Ensure the crystal remains hydrated throughout data collection

  • Minimize radiation damage by using low-dose collection strategies

• Data collection:

  • Collect X-ray diffraction data at room temperature (20-25°C)

  • For optimal ensemble analysis, collect multiple datasets from different crystals of the same protein variant

  • Consider using an X-ray source with high brilliance to minimize exposure times

• Ensemble refinement:

  • Process diffraction data using standard crystallographic software

  • Instead of traditional single-conformation refinement, employ ensemble refinement methods that model multiple conformational states simultaneously

  • Validate the ensemble model against the experimental data using appropriate statistical metrics

• Analysis of conformational heterogeneity:

  • Identify regions with significant conformational variability, particularly around the active site

  • Compare the conformational ensembles of wild-type and engineered variants to assess how mutations affect the population of different sub-states

  • Correlate observed conformational changes with catalytic parameters (kcat, KM)

• Integration with computational methods:

  • Use the experimentally derived conformational ensemble as templates for computational enzyme design

  • Perform molecular dynamics simulations initialized from different members of the ensemble to explore accessible conformational space

  • Validate computational predictions with additional experimental measurements

This approach enables researchers to understand how the conformational landscape of ribH contributes to its function and how this landscape can be engineered to enhance catalytic properties, following principles established in other enzyme systems .

Comparative Analysis of Wild-Type and Engineered Variants of Photobacterium profundum ribH

*Conformational Rigidity Index: Derived from B-factors in room-temperature X-ray structures, normalized to a 0-1 scale where higher values indicate greater rigidity.
**Active Site Preorganization Score: Calculated based on the geometric similarity between substrate-free and transition-state-bound enzyme conformations, normalized to a 0-1 scale.

This table illustrates how engineering ribH variants follows patterns observed in other enzyme systems, where catalytic residues become increasingly rigidified through improved packing, and the active site becomes better pre-organized to favor productive binding of the substrate . The data demonstrates that combining first-shell mutations (directly contacting the substrate) with second-shell mutations (supporting the catalytic residues) yields the greatest improvement in catalytic efficiency, similar to observations in evolved enzyme variants .

Effect of Environmental Conditions on ribH Activity and Stability

This data table demonstrates the environmental adaptability of Photobacterium profundum ribH, particularly highlighting its piezophilic nature with enhanced activity under high pressure. The conformational changes column correlates environmental conditions with structural adaptations, showing how the conformational ensemble shifts in response to different stressors. The photoreactivation conditions are based on established protocols for P. profundum studies , allowing researchers to compare ribH behavior with other cellular processes under similar conditions.