Recombinant Prochlorococcus marinus subsp. pastoris 6,7-dimethyl-8-ribityllumazine synthase (ribH)

What is the basic structure and function of 6,7-dimethyl-8-ribityllumazine synthase (ribH) in Prochlorococcus marinus?

6,7-dimethyl-8-ribityllumazine synthase (ribH) in Prochlorococcus marinus is a critical enzyme in the riboflavin biosynthetic pathway. Structurally, ribH typically forms a homopentameric structure with subunits of approximately 17 kDa, similar to the enzyme found in other species like Schizosaccharomyces pombe. Functionally, it catalyzes 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, which is the penultimate step in riboflavin biosynthesis .

Unlike some bacterial lumazine synthases that form icosahedral capsids (532-symmetric hollow capsids described as dodecamers of pentamers), the P. marinus enzyme is believed to function as a homopentamer with C5-symmetry. The active sites are located at the interfaces between adjacent subunits in the pentamer, a feature conserved across species .

How does the ribH gene organization in Prochlorococcus marinus compare to other cyanobacteria?

In Prochlorococcus marinus, the ribH gene is part of the riboflavin biosynthetic pathway genes organized in the genome. While some bacteria have ribH genes located within a riboflavin operon alongside other riboflavin biosynthesis genes, the exact organization in P. marinus can be compared to other cyanobacteria.

For context, in organisms like Brucella species, there are two genes (ribH1 and ribH2) located on different chromosomes, with ribH1 located inside a small riboflavin operon, together with two other riboflavin biosynthesis genes and the nusB gene, which specifies an antitermination factor . In P. marinus, the genomic organization reflects its streamlined genome (approximately 1.66 Mb) with relatively few paralogous genes compared to other cyanobacteria, as shown in this comparative data:

The genomic context of ribH is significant for understanding its regulation and potential co-expression with other genes involved in riboflavin biosynthesis or related metabolic pathways .

What are the optimal conditions for expressing recombinant Prochlorococcus marinus ribH in E. coli?

For optimal expression of recombinant P. marinus ribH in E. coli, researchers should consider the following methodological approach:

• Vector selection: Use a T7 promoter-based expression vector (such as pT7-7) that allows for tight regulation and high-level expression.

• E. coli strain: BL21(DE3) or similar strains are recommended due to their lack of proteases and compatibility with T7 promoter systems.

• Codon optimization: Consider codon optimization for E. coli, as P. marinus has an AT-rich genome (30.8% GC content) which differs from E. coli's codon usage .

• Expression conditions: Culture at 30°C rather than 37°C after induction to enhance protein folding and solubility. IPTG concentration of 0.1-0.5 mM is typically sufficient.

• Media supplementation: Supplement with riboflavin (10 μM) to stabilize the recombinant protein.

Taking cues from similar ribH expressions, like that done with Schizosaccharomyces pombe ribH:

• Express using recombinant E. coli strains

• Expect expression yields of homopentameric protein with subunits of approximately 17 kDa

• Anticipate an apparent molecular mass of about 85-90 kDa as determined by sedimentation equilibrium centrifugation (with sedimentation at approximately 5.0 S at 20°C)

What purification strategy yields the highest activity for recombinant P. marinus ribH?

A multi-step purification strategy is recommended to obtain pure, highly active recombinant P. marinus ribH:

• Initial clarification: After cell lysis (usually by sonication or pressure homogenization), centrifuge at 15,000-20,000 × g for 30 minutes to remove cell debris.

• Ammonium sulfate fractionation: Perform stepwise precipitation, with ribH typically precipitating at 45-60% saturation.

• Ion exchange chromatography: Apply to a DEAE-Sepharose column equilibrated with buffer (typically 50 mM Tris-HCl, pH 7.5-8.0), and elute with a gradient of 0-0.5 M NaCl.

• Size exclusion chromatography: Further purify using a Superdex 200 column to separate the pentameric ribH from monomers and other contaminants.

• Storage conditions: Store in 50 mM potassium phosphate buffer (pH 7.0) containing 1 mM DTT and 1 mM EDTA with 10% glycerol at -80°C to maintain activity.

This protocol typically yields protein with specific activity comparable to other recombinant lumazine synthases. Based on analogous enzymes, expected activity levels would be around 10,000-15,000 nmol·mg⁻¹·h⁻¹, with Km values of approximately 5 μM for 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 60-70 μM for 3,4-dihydroxy-2-butanone 4-phosphate .

How can I accurately measure the kinetic parameters of recombinant P. marinus ribH?

To accurately measure kinetic parameters of recombinant P. marinus ribH, employ the following methodology:

• Assay principle: The enzymatic reaction forms 6,7-dimethyl-8-ribityllumazine, which has characteristic absorption and fluorescence properties that can be measured spectrophotometrically.

• Standard reaction conditions:

  • Buffer: 100 mM potassium phosphate, pH 7.0

  • Temperature: 37°C (physiologically relevant) or 30°C (for comparison with other studies)

  • Substrate ranges:

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

    • 3,4-dihydroxy-2-butanone 4-phosphate: 5-500 μM

• Measurement approaches:

  • Spectrophotometric monitoring at 408-410 nm for product formation

  • HPLC analysis for direct quantification of substrate consumption and product formation

  • Fluorescence-based assays (excitation: 410 nm, emission: 490 nm)

• Data analysis: For accurate determination, use appropriate enzyme kinetic models:

  • Michaelis-Menten equation for simple kinetics

  • Hill equation if cooperativity is observed (as seen with other lumazine synthases)

Expected kinetic parameters based on similar enzymes:

• vmax: 10,000-20,000 nmol·mg⁻¹·h⁻¹

• Km for 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione: 1-10 μM

• Km for 3,4-dihydroxy-2-butanone 4-phosphate: 50-150 μM

For cooperative binding analysis, fit data to the Hill equation:
V = Vmax × S^n / (K^n + S^n)
where V is reaction velocity, Vmax is maximum velocity, S is substrate concentration, K is the reaction constant, and n is the Hill coefficient .

What experimental approaches can determine the oligomeric state and structural properties of P. marinus ribH?

To determine the oligomeric state and structural properties of P. marinus ribH, implement multiple complementary experimental approaches:

When applying these methods to P. marinus ribH, researchers should anticipate pentameric assembly with structural features similar to other lumazine synthases, including active sites at subunit interfaces and binding sites for substrate molecules and riboflavin .

What strategies can be employed to improve the catalytic efficiency of P. marinus ribH through site-directed mutagenesis?

To improve the catalytic efficiency of P. marinus ribH through site-directed mutagenesis, consider the following structured approach:

• Target residue identification:

  • Focus on active site residues identified through structural analysis or homology modeling

  • Key conserved residues in lumazine synthases include residues equivalent to Trp27 in homologous enzymes, which significantly impact substrate binding and riboflavin interaction

  • Target residues at the subunit interfaces where the active sites are located

• Rational mutation design based on mechanistic insights:

  • Enhance substrate binding by introducing residues with higher affinity for substrate molecules

  • Improve transition state stabilization through introduction of polarizing groups

  • Modify the microenvironment of the active site to optimize proton transfer reactions

• Methodological approach:

  • Use overlap extension PCR or QuikChange mutagenesis for introducing mutations

  • Create a library of variants with single, double, or combinatorial mutations

  • Express mutants in E. coli and compare activity with wild-type enzyme

• Evaluating effects of mutations:

  • Measure kinetic parameters (kcat, Km, kcat/Km) for each variant

  • Assess structural integrity through thermal stability assays

  • Determine substrate specificity changes

Drawing from studies of homologous enzymes, the following mutations might be considered:

• Tryptophan to tyrosine/phenylalanine at the position equivalent to Trp27, which may alter riboflavin binding without significantly affecting kinetic properties

• Replacement of active site tryptophan with aliphatic amino acids, which might reduce affinity for both riboflavin and substrate

How can the Prochlorococcus marinus genome cloning in yeast be leveraged for ribH engineering?

The successful cloning of the entire Prochlorococcus marinus MED4 genome (1.66 Mb) in Saccharomyces cerevisiae provides a powerful platform for ribH engineering through these methodological approaches:

• Yeast-based genome engineering strategy:

  • Use the cloned P. marinus genome in yeast for targeted modifications of ribH

  • Exploit yeast's efficient homologous recombination machinery

  • Follow protocols similar to those used for Mycoplasma genome engineering in yeast

• Technical implementation:

  • Design targeting cassettes with homology arms flanking the ribH gene

  • Introduce modified ribH variants through transformation of linear DNA fragments

  • Select transformants using appropriate markers

  • Verify genome modifications by PCR and sequencing

• Advantages of this approach:

  • Enables engineering in the context of the complete P. marinus genome

  • Allows for evaluation of ribH modifications in relation to other genomic elements

  • Provides a platform for testing ribH variants before attempting more challenging genetic manipulation in P. marinus itself

• Considerations for successful implementation:

  • Account for the AT-rich nature of the P. marinus genome (30.8% GC content)

  • Utilize abundant yeast replication origin consensus sites naturally present in the P. marinus genome

  • Be aware of potential mutations that might arise during yeast propagation (previous studies identified single base pair missense mutations, frameshifts, and other alterations)

This approach leverages the remarkable achievement of maintaining the intact 1.66 Mb bacterial genome in S. cerevisiae, creating opportunities for comprehensive genetic engineering of ribH and related genes.

What are the current applications of P. marinus ribH in biotechnology and drug discovery?

P. marinus ribH has several significant applications in biotechnology and drug discovery, particularly leveraging its structural properties and essential role in metabolism:

• Drug target development:

  • Similar to lumazine synthase (ribH) in Mycobacterium tuberculosis, P. marinus ribH represents a potential target for antimicrobial development

  • High-throughput screening approaches can identify inhibitors through molecular docking of compound libraries (as demonstrated with M. tuberculosis ribH where ~600,000 compounds were screened)

  • The essentiality of ribH for bacterial survival makes it an attractive target for antibiotic development against resistant marine pathogens

• Protein engineering applications:

  • The self-assembling nature of lumazine synthases makes them valuable scaffolds for:

    • Enzyme immobilization

    • Vaccine development (presentation of antigens on multivalent particles)

    • Nanoreactor design (encapsulation of enzymes within protein cages)

• Biosensor development:

  • ribH's interaction with riboflavin can be exploited for the development of biosensors

  • The fluorescence quenching observed when riboflavin binds to lumazine synthase (with quantum yield <2% compared to free riboflavin) provides a mechanism for detection systems

• Biomarker potential:

  • The 6,7-dimethyl-8-ribityllumazine produced by ribH serves as a MAIT cell-activating ligand, offering potential applications in immunomodulation

  • This connection to immune response opens avenues for diagnostic or therapeutic approaches

• Current research focus:

  • Structure-based drug design targeting ribH has yielded promising compounds with antimicrobial activity

  • In studies with M. tuberculosis ribH, identified inhibitors showed potent activity against intracellular bacteria and synergistic effects with first-line antibiotics

These applications are supported by the detailed characterization of ribH structure, function, and essentiality across various bacterial species.

What statistical methods are most appropriate for analyzing ribH enzyme kinetics data?

For robust analysis of ribH enzyme kinetics data, appropriate statistical methods should be selected based on the specific experimental design and data characteristics:

• For basic kinetic parameter estimation:

  • Non-linear regression using the Michaelis-Menten equation or Hill equation for cooperative binding

  • Report parameters with confidence intervals rather than just point estimates

  • Calculate standard errors for Km, Vmax, and kcat values using appropriate error propagation

• For comparing kinetic parameters between wild-type and mutant enzymes:

  • For normally distributed data: t-tests (two conditions) or one-way ANOVA (multiple conditions)

  • For non-normally distributed data: Mann-Whitney U test (two conditions) or Kruskal-Wallis test (multiple conditions)

  • Post-hoc analysis for multiple comparisons (e.g., Tukey's HSD or Bonferroni correction)

• For inhibition studies:

  • Global fitting of different inhibition models (competitive, non-competitive, uncompetitive)

  • Akaike Information Criterion (AIC) to select the best-fitting model

  • Bootstrap resampling for robust parameter estimation and confidence intervals

• For thermal stability and pH dependency studies:

  • Non-linear regression using sigmoidal dose-response curves

  • Determination of inflection points (Tm, pKa) with appropriate confidence intervals

The statistical analysis should adhere to these key principles:

• Report descriptive statistics (mean, standard deviation, median, interquartile range)

• Use appropriate parametric or non-parametric methods based on data distribution

• Apply multiple comparison corrections when testing multiple hypotheses

• Consider experimental variability and sample size in interpretation

How can single-subject experimental design be applied to study inhibitors of P. marinus ribH?

Single-subject experimental design (SSED) can be effectively applied to study inhibitors of P. marinus ribH by focusing on detailed analysis of individual inhibitor compounds rather than group averages, particularly useful when screening novel inhibitors:

• SSED application to ribH inhibitor studies:

  • Implement baseline-intervention designs where each potential inhibitor serves as its own control

  • Conduct intensive repeated measurements of inhibition at varying concentrations

  • Track changes in level, trend, and variability of enzyme activity

• Experimental implementation:

  • A-B-A-B withdrawal design: measure enzyme activity without inhibitor (A), with inhibitor (B), after inhibitor removal (A), and upon reintroduction (B)

  • Multiple baseline design: stagger introduction of inhibitor across different reaction conditions

  • Changing criterion design: systematically adjust inhibitor concentration to establish dose-response relationships

• Data visualization and analysis approaches:

  • Time-series graphs to visualize enzyme activity across phases

  • Visual analysis of level, trend, and variability changes (as illustrated in Figure 1, Panel B from search result )

  • Calculation of effect sizes specific to single-subject designs (e.g., percentage of non-overlapping data, Tau-U)

• Advantages for ribH inhibitor research:

  • Detailed characterization of individual inhibitors without averaging effects

  • Flexibility to adjust experimental parameters based on ongoing results

  • Ability to detect unique inhibition mechanisms that might be masked in group designs

  • Efficient use of resources when testing novel compounds with limited availability

• Integration with standard biochemical approaches:

  • Determine IC50 values through dose-response curves for each inhibitor

  • Characterize inhibition mechanisms (competitive, non-competitive, etc.)

  • Complement with structural studies to understand binding modes

This approach is particularly valuable for initial characterization of novel inhibitors before proceeding to more resource-intensive group-based experimental designs .

How can advanced sequence analysis techniques be applied to study evolution of ribH across marine cyanobacteria?

Advanced sequence analysis techniques can provide comprehensive insights into the evolution of ribH across marine cyanobacteria through the following methodological framework:

• Comprehensive sequence acquisition and alignment:

  • Collect ribH sequences from diverse marine cyanobacteria, including multiple Prochlorococcus ecotypes

  • Implement profile-based multiple sequence alignment using MAFFT or similar algorithms

  • Refine alignments based on structural information when available

• Phylogenetic reconstruction methods:

  • Maximum likelihood analysis using models that account for site-specific rate variation

  • Bayesian inference to obtain posterior probability distributions for evolutionary relationships

  • Gene tree-species tree reconciliation to identify potential horizontal gene transfer events

• Selection pressure analysis:

  • Calculate dN/dS ratios to identify sites under purifying or positive selection

  • Implement branch-site models to detect episodic selection in specific lineages

  • Use mixed effects model of evolution (MEME) to detect episodic diversifying selection

• Structural evolution analysis:

  • Map sequence conservation onto structural models to identify functionally constrained regions

  • Predict the effects of amino acid substitutions on protein stability and function

  • Analyze co-evolution patterns to identify functionally linked residue networks

• Ecological correlation analysis:

  • Correlate sequence features with ecological parameters (depth, temperature, light intensity)

  • Compare high-light and low-light adapted Prochlorococcus ecotypes

  • Analyze ribH evolution in the context of genome streamlining in Prochlorococcus

This approach leverages the information that P. marinus exists in different ecological forms (high-light-adapted genotypes in upper waters and low-light-adapted genotypes at the bottom of the illuminated layer) and has undergone genome streamlining with specific adaptations .

How can I troubleshoot low activity or instability issues with recombinant P. marinus ribH?

When encountering low activity or instability with recombinant P. marinus ribH, implement this systematic troubleshooting approach:

• Expression optimization:

  • Test multiple E. coli expression strains (BL21(DE3), Rosetta, Arctic Express)

  • Optimize induction conditions: lower IPTG concentration (0.1-0.2 mM) and reduced temperature (16-20°C)

  • Consider co-expression with chaperones to improve folding

  • Use auto-induction media for gentler protein expression

• Protein solubility enhancement:

  • Add solubility tags (MBP, SUMO, TrxA) to improve folding and solubility

  • Test different lysis buffers with various additives:

    • Glycerol (10-20%)

    • Non-ionic detergents (0.1% Triton X-100)

    • Stabilizing agents (1 mM DTT, 5 mM β-mercaptoethanol)

  • Include riboflavin (10-50 μM) in all buffers to stabilize the enzyme

• Activity preservation strategies:

  • Supplement purification buffers with cofactors (e.g., Mg²⁺ at 5-10 mM)

  • Minimize exposure to freeze-thaw cycles

  • Store enzyme with glycerol (20-30%) at -80°C in small aliquots

  • Test enzyme activity immediately after purification to establish baseline

• Enzyme assay optimization:

  • Verify substrate quality and preparation

  • Test multiple buffer systems (phosphate, Tris, HEPES) at different pH values

  • Optimize assay temperature (30-40°C range)

  • Include BSA (0.1-1 mg/ml) to prevent surface adsorption

• Activity rescue strategies:

  • Attempt partial refolding if inclusion bodies form

  • Test different pentamer stabilization conditions

  • Consider on-column refolding during purification

Based on studies with similar enzymes, remember that ribH activity is highly dependent on Mg²⁺ concentration and temperature, with activity typically increasing at higher temperatures, possibly due to relaxation of complex tertiary structures into more extended states that facilitate substrate binding .

What are the key considerations when designing experiments to compare wild-type and mutant versions of P. marinus ribH?

When designing experiments to compare wild-type and mutant versions of P. marinus ribH, consider these critical methodological factors to ensure valid and reproducible results:

• Experimental design principles:

  • Include appropriate controls in every experiment (wild-type, negative controls, known mutants)

  • Use paired experimental designs whenever possible to minimize batch effects

  • Implement biological replicates (minimum n=3) for each construct

  • Randomize sample processing order to avoid systematic biases

• Protein expression and purification considerations:

  • Express all variants under identical conditions

  • Purify proteins in parallel using the same protocol

  • Verify protein purity by SDS-PAGE (aim for >95% purity)

  • Quantify protein concentration using multiple methods (Bradford/BCA and UV absorbance)

  • Confirm pentameric assembly by size exclusion chromatography or native PAGE

• Structural and stability assessment:

  • Perform circular dichroism to compare secondary structure content

  • Conduct thermal shift assays to assess folding stability

  • If possible, obtain crystal structures of key mutants

• Activity comparison methodology:

  • Test enzymatic activity under multiple conditions:

    • Various substrate concentrations

    • Temperature range (25-45°C)

    • Different Mg²⁺ concentrations (1-10 mM)

  • Determine full kinetic parameters rather than single-point activity measurements

  • Consider effects of mutations on both substrates of the reaction

• Data analysis and reporting standards:

  • Report all experimental conditions in detail

  • Present enzyme kinetic data in tabular format with statistical analysis

  • Include representative raw data plots along with processed results

An example data presentation format, modeled after rigorous enzyme characterization studies:

This approach ensures meaningful comparisons between wild-type and mutant enzymes while accounting for experimental variability .

What emerging technologies could advance our understanding of P. marinus ribH function and applications?

Several cutting-edge technologies are poised to significantly advance our understanding of P. marinus ribH function and expand its applications:

• Cryo-electron microscopy (Cryo-EM):

  • High-resolution structural determination without crystallization

  • Visualization of conformational changes during catalysis

  • Structure-based drug design targeting specific conformational states

  • Potential to resolve heterogeneous oligomeric states in solution

• Single-molecule enzymology:

  • Direct observation of ribH catalytic cycle through fluorescence resonance energy transfer (FRET)

  • Characterization of conformational dynamics during substrate binding and product release

  • Understanding cooperativity mechanisms at the single-molecule level

  • Detection of rare or transient catalytic states

• In-cell structural biology:

  • NMR spectroscopy of isotopically labeled ribH in living cells

  • Mass spectrometry-based footprinting to map protein-protein interactions in vivo

  • Visualization of ribH localization and dynamics using super-resolution microscopy

• Synthetic biology applications:

  • Engineering ribH-based protein cages for targeted drug delivery

  • Development of ribH-based biosensors for environmental monitoring

  • Creation of artificial metabolic pathways incorporating modified ribH variants

  • Protein scaffold engineering for multi-enzyme cascade reactions

• Computational advances:

  • Machine learning approaches for predicting ribH mutant properties

  • Molecular dynamics simulations at extended timescales to capture complete catalytic cycles

  • Quantum mechanics/molecular mechanics (QM/MM) studies of the reaction mechanism

  • Network analysis of ribH's position in metabolic and regulatory networks

• Genome engineering:

  • CRISPR-interference (CRISPRi) for conditional knockdown studies in P. marinus

  • Whole-cell mutational analysis to understand ribH's role in cellular physiology

  • Application of the yeast-based P. marinus genome cloning system for comprehensive genetic engineering

These emerging technologies will enable researchers to bridge the gaps between molecular mechanism, cellular function, and biotechnological applications of P. marinus ribH.

How might climate change impact the evolution and function of ribH in marine cyanobacteria like Prochlorococcus?

Climate change may significantly impact the evolution and function of ribH in marine cyanobacteria through multiple mechanisms that merit investigation:

• Thermal adaptation mechanisms:

  • Rising ocean temperatures may select for ribH variants with altered thermostability

  • Research approach: Compare ribH sequences and enzyme activities across Prochlorococcus strains from different temperature regimes

  • Methodology: Thermal shift assays and enzyme kinetics at variable temperatures to identify adaptive changes

  • Hypothesis: Thermally adapted variants may show shifts in optimal temperature for activity at the expense of catalytic efficiency

• Light intensity adaptations:

  • Changes in ocean stratification affecting light penetration may impact ribH evolution

  • Research approach: Compare ribH from high-light vs. low-light adapted Prochlorococcus ecotypes

  • Methodology: Characterize ribH expression and activity under simulated future light conditions

  • Hypothesis: Different selective pressures may act on ribH in surface vs. deep-water ecotypes

• Ocean acidification effects:

  • Decreasing pH may alter the ionization state of key catalytic residues

  • Research approach: Characterize ribH activity across pH gradients reflecting future ocean conditions

  • Methodology: pH-dependent enzyme kinetics and structural studies

  • Hypothesis: acidification may select for pH-robust variants with altered catalytic mechanisms

• Nutrient limitation responses:

  • Changes in nutrient availability may affect riboflavin biosynthesis regulation

  • Research approach: Examine ribH expression under nutrient-limited conditions mimicking future scenarios

  • Methodology: Transcriptomics and metabolomics under simulated future conditions

  • Hypothesis: Nutrient limitation may drive more efficient riboflavin pathway regulation

• Community interaction shifts:

  • Altered marine microbiome composition may impact ribH evolution through changing selection pressures

  • Research approach: Study horizontal gene transfer patterns of ribH among marine microorganisms

  • Methodology: Comparative genomics across time-series samples from ocean observatories

  • Hypothesis: Increased frequency of extreme events may accelerate ribH gene transfer and evolution