Recombinant Prochlorococcus marinus subsp. pastoris Aspartate carbamoyltransferase (pyrB)

What is Aspartate carbamoyltransferase (pyrB) and what is its function in Prochlorococcus marinus?

Aspartate carbamoyltransferase (ATCase, EC 2.1.3.2) is a key enzyme in the pyrimidine biosynthetic pathway, catalyzing the condensation of carbamoyl phosphate with L-aspartate to form N-carbamoyl-L-aspartate. In Prochlorococcus marinus, this enzyme plays a critical role in nucleotide metabolism and is essential for cellular growth and replication .

How does Prochlorococcus marinus subsp. pastoris differ from other strains in the Prochlorococcus genus?

Prochlorococcus marinus subsp. pastoris (strain CCMP1986/NIES-2087/MED4) is a high-light-adapted ecotype isolated from the surface waters (5m depth) of the Mediterranean Sea . Key differences include:

This strain is particularly valuable for research due to its compact genome and adaptation to high-light environments, representing one of the smallest genomes of a photosynthetic organism .

What are the optimal storage and handling conditions for recombinant pyrB protein?

For optimal stability and activity of recombinant Prochlorococcus marinus subsp. pastoris pyrB protein:

• Storage Temperature: Store at -20°C/-80°C. The shelf life of liquid preparations is typically 6 months, while lyophilized forms can remain stable for up to 12 months .

• Reconstitution Protocol:

  • Briefly centrifuge the vial to collect contents at the bottom

  • Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Add glycerol to a final concentration of 5-50% (50% is recommended) for long-term storage

  • Aliquot for storage to avoid repeated freeze-thaw cycles

• Working Aliquots: For short-term experiments, store working aliquots at 4°C for up to one week, as repeated freezing and thawing significantly reduces enzyme activity .

• Buffer Considerations: The stability is affected by buffer components, storage temperature, and the intrinsic properties of the protein itself .

How can I express and purify recombinant Prochlorococcus marinus pyrB for functional studies?

Expression and purification of recombinant pyrB protein requires careful consideration of host systems, expression conditions, and purification strategies:

Expression System Selection:
E. coli is commonly used as a heterologous host for pyrB expression due to its well-established genetic tools and rapid growth . While numerous E. coli strains can be used, BL21(DE3) derivatives are often preferred for their reduced protease activity and ability to express toxic proteins.

Expression Protocol:

• Clone the full-length pyrB gene (encoding amino acids 1-338) into an appropriate expression vector containing an inducible promoter (e.g., T7) .

• Transform into competent E. coli cells and select transformants on appropriate selective media.

• Optimize expression conditions: typically, induction at OD600 0.6-0.8 with IPTG concentrations between 0.1-1.0 mM at 16-30°C for 4-18 hours provides the best balance between yield and solubility.

• Harvest cells by centrifugation and lyse using mechanical disruption or chemical methods.

Purification Strategy:
Most recombinant pyrB proteins can be purified using affinity chromatography if tagged (His, GST, etc.), followed by size exclusion chromatography to obtain highly pure protein . For Prochlorococcus marinus pyrB specifically, a purity of >85% can be achieved using SDS-PAGE analysis .

Activity Verification:
Measure enzymatic activity using a spectrophotometric assay monitoring the formation of N-carbamoyl-L-aspartate from carbamoyl phosphate and L-aspartate. This is critical to ensure that the recombinant protein maintains its native function .

What methods can be used to study the structural features of Prochlorococcus marinus pyrB?

Several complementary approaches can be employed to elucidate the structural features of Prochlorococcus marinus pyrB:

Sequence-Based Analysis:

• Multiple sequence alignment with other known ATCase proteins, particularly the well-characterized E. coli enzyme, can reveal conserved catalytic and regulatory domains .

• The full sequence of the protein (338 amino acids) contains key domains that can be identified through bioinformatic analysis .

Experimental Structural Studies:

• X-ray Crystallography: The highest resolution method for determining the three-dimensional structure of the protein. This requires:

  • Production of highly pure (>95%) protein

  • Screening of crystallization conditions

  • Diffraction data collection and structure determination

• Small-Angle X-ray Scattering (SAXS): Useful for studying the quaternary structure in solution and conformational changes upon substrate or regulator binding.

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): Provides information about solvent accessibility and conformational dynamics.

• Circular Dichroism (CD): For assessment of secondary structure content and thermal stability.

Enzymatic and Binding Studies:

• Substrate binding kinetics using isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR).

• Analysis of homotropic (substrate) and heterotropic (allosteric) effects on enzyme activity .

These methods can reveal similarities and differences between Prochlorococcus marinus pyrB and other bacterial ATCases, potentially providing insights into environmental adaptations.

How do regulatory requirements differ for experiments involving recombinant Prochlorococcus marinus proteins?

Experiments involving recombinant Prochlorococcus marinus proteins are subject to various regulatory frameworks, particularly in academic and research settings:

NIH Guidelines Applicability:
According to the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules, many experiments involving recombinant Prochlorococcus proteins may be exempt under Section III-F, provided they meet specific criteria :

• The recombinant nucleic acids are not designed to integrate into DNA.

• They don't produce toxins lethal to vertebrates at an LD50 of less than 100 nanograms per kilogram body weight.

• They don't involve whole plant regeneration or large-scale cultures (>10 liters).

Important Regulatory Considerations:

• While the expressed proteins themselves (such as recombinant pyrB) are not subject to the NIH Guidelines, the nucleic acid constructs used to produce them are regulated .

• Experiments involving the introduction of recombinant DNA into Risk Group 2 agents typically require IBC (Institutional Biosafety Committee) review, though exceptions exist for host-specific propagation .

• Tissue culture experiments with recombinant nucleic acids may be exempt, but this exemption doesn't automatically extend to all experiments with derived cell lines .

Documentation Requirements:
Researchers should maintain records documenting:

• Risk assessment for the specific recombinant construct

• Containment measures appropriate for the risk level

• Approval from relevant institutional committees when required

These requirements ensure responsible research while minimizing unnecessary regulatory burden for low-risk recombinant protein work.

How can comparative genomic analysis of pyrB genes inform our understanding of Prochlorococcus evolution and adaptation?

Comparative genomic analysis of pyrB genes across Prochlorococcus ecotypes provides valuable insights into evolutionary adaptations to different oceanographic niches:

Methodology for Comparative Analysis:

• Obtain pyrB sequences from diverse Prochlorococcus strains through whole genome sequencing or targeted gene amplification.

• Perform phylogenetic analysis using maximum likelihood or Bayesian methods to establish evolutionary relationships.

• Calculate selection metrics (dN/dS ratios) to identify regions under positive, neutral, or purifying selection.

• Map sequence variations to structural models to assess potential functional implications.

Key Findings from Existing Analyses:
Genomic studies of Prochlorococcus strains have revealed that pyrB is part of the core genome shared across all ecotypes, despite significant genomic streamlining . The average Prochlorococcus genome contains approximately 2,000 genes, with 1,273 genes common to all strains . This conservation highlights the essential nature of pyrimidine metabolism for survival.

Ecological Implications:
The sequence variation in pyrB and other metabolic genes correlates with adaptation to different light regimes, nutrient availability, and ocean depths . High-light adapted ecotypes like MED4 show genomic adaptations for surface water environments where light is abundant but nutrients may be scarce, while low-light adapted strains like SS120 display adaptations for deeper, more nutrient-rich waters with limited light penetration .

Through such comparative analyses, researchers can identify key mutations that may have facilitated the remarkable diversification and ecological success of Prochlorococcus across the world's oceans.

What mechanisms regulate pyrB expression in Prochlorococcus marinus, and how do they differ from other bacterial systems?

The regulation of pyrB expression in Prochlorococcus marinus reflects unique adaptations to its oligotrophic marine environment, with several notable differences from model systems like E. coli:

Regulatory Mechanisms in Model Bacterial Systems:
In E. coli and related enterobacteria, pyrB is typically regulated through:

• A bicistronic operon structure (pyrBI) with coordinated expression of catalytic and regulatory subunits .

• Transcriptional attenuation involving a leader peptide sequence .

• Negative feedback regulation by pyrimidine nucleotides, particularly UTP .

• Translational coupling between pyrB and pyrI, ensuring stoichiometric production of both subunits .

Prochlorococcus-Specific Regulatory Features:
Genomic and transcriptomic analyses suggest several unique aspects of pyrB regulation in Prochlorococcus:

• Genomic Context: While the pyrBI operon structure appears conserved, the intergenic regions and surrounding genomic context may differ from E. coli .

• Transcriptional Regulation: Transcription start sites in Prochlorococcus MED4 have been predicted using raster-score filter methods, accounting for the higher G+C content (34%) within upstream regions compared to E. coli .

• Light-Dependent Regulation: Unlike many bacterial regulatory systems, Prochlorococcus gene expression is often synchronized with light-dark cycles, suggesting potential light-dependent regulation of basic metabolic pathways including pyrimidine synthesis .

• Streamlined Regulatory Networks: With its minimal genome, Prochlorococcus has likely eliminated redundant regulatory mechanisms, potentially relying on fewer transcription factors and simpler feedback systems .

Experimental Approaches to Study Regulation:

• Reporter gene assays using promoter-GFP fusions to quantify expression under different environmental conditions.

• RNA-seq analysis to identify co-regulated genes and potential operons.

• Chromatin immunoprecipitation (ChIP) to identify regulatory protein binding sites.

• In vitro transcription and translation assays to directly measure regulatory effects.

These regulatory adaptations likely contribute to the ecological success of Prochlorococcus in nutrient-limited oceanic environments.

How does the catalytic mechanism of Prochlorococcus marinus pyrB compare to that of other organisms, and what implications does this have for enzyme engineering?

The catalytic mechanism of Prochlorococcus marinus pyrB shows both conservation of essential features and unique adaptations that provide insights for enzyme engineering:

Conserved Catalytic Mechanism:
The fundamental reaction catalyzed by ATCase involves several key steps:

• Binding of carbamoyl phosphate to form a carbamoyl-enzyme intermediate

• Nucleophilic attack by the amino group of aspartate

• Formation of N-carbamoyl-L-aspartate and release of inorganic phosphate

The catalytic residues involved in this mechanism are highly conserved across species, from E. coli to archaeal organisms like Sulfolobus acidocaldarius .

Structural and Functional Comparisons:

Implications for Enzyme Engineering:
The unique properties of Prochlorococcus pyrB offer several opportunities for enzyme engineering:

• Environmental Adaptation: Understanding how Prochlorococcus ATCase has adapted to function in oligotrophic marine environments could inform the design of enzymes for specific environmental conditions.

• Minimal Functional Core: The streamlined genome of Prochlorococcus suggests its ATCase may represent a minimal functional version of the enzyme, identifying essential residues for catalysis.

• Novel Regulatory Mechanisms: Any unique regulatory features could be exploited to create synthetic enzymes with custom regulatory properties.

• Structure-Based Engineering: Comparing the structures of ATCase from diverse organisms helps identify regions amenable to modification without disrupting catalytic function.

• Application Potential: Engineered variants could find applications in biosensors, biocatalysis for pharmaceutical precursor synthesis, or as model systems for studying allosteric regulation.

To fully realize this potential, further structural and biochemical characterization of Prochlorococcus marinus pyrB is needed to identify its unique adaptations and catalytic properties.

How do environmental factors affect the stability and activity of Prochlorococcus marinus pyrB?

As a protein from a marine cyanobacterium that inhabits specific ecological niches, Prochlorococcus marinus pyrB has evolved to function optimally under particular environmental conditions:

Temperature Effects:
Prochlorococcus marinus MED4 (subsp. pastoris) thrives in surface waters of tropical and subtropical oceans , suggesting that its enzymes, including pyrB, are adapted to function optimally around 20-25°C. While not as thermostable as enzymes from thermophilic organisms like Sulfolobus acidocaldarius , it likely exhibits reasonable stability within its physiological temperature range.

Experimental approach to determine thermal stability:

• Differential scanning calorimetry (DSC) to determine melting temperature (Tm)

• Activity assays at various temperatures to establish the temperature optimum

• Thermal inactivation kinetics to assess stability over time at elevated temperatures

pH Dependence:
The cytoplasmic pH of cyanobacteria is typically maintained around 7.0-7.5, suggesting that Prochlorococcus pyrB would function optimally in this range. Experimental determination of pH optima is essential for accurate activity measurements.

Light and Oxygen Responsiveness:
Recent studies on Prochlorococcus marinus responses to light and oxygen have revealed significant physiological adaptations . The high-light adapted MED4 strain was isolated from surface waters where oxygen concentration is near saturation , suggesting that its enzymes function well under aerobic conditions. While pyrB itself may not be directly light-regulated, its activity could be indirectly affected by light-driven changes in cellular metabolism and energy status.

Salt Concentration Effects:
As a marine organism, Prochlorococcus enzymes are adapted to function in the presence of relatively high salt concentrations (~3.5% in seawater). Recombinant pyrB may exhibit optimal activity and stability in buffers that mimic these ionic conditions.

Experimental protocol to assess salt effects:

• Measure enzyme activity across a range of NaCl concentrations (0-1M)

• Determine protein stability via thermal shift assays at various salt concentrations

• Assess structural changes using circular dichroism spectroscopy

Understanding these environmental dependencies is crucial for both accurate in vitro characterization and potential biotechnological applications of the enzyme.

What techniques can be used to assess pyrB enzymatic activity, and how do kinetic parameters compare across species?

Multiple complementary techniques can be employed to assess pyrB enzymatic activity, each with specific advantages:

Spectrophotometric Assays:

• Direct Colorimetric Assay: Measures the formation of N-carbamoyl-L-aspartate using colorimetric reagents that react with the ureido group.

• Coupled Enzyme Assays: Links ATCase activity to the production of a chromophore or fluorophore through secondary enzymatic reactions.

Radiochemical Assays:
Using radiolabeled substrates (14C-aspartate or 14C-carbamoyl phosphate) to track product formation with high sensitivity.

Direct Product Quantification:
HPLC or LC-MS based methods to directly quantify N-carbamoyl-L-aspartate formation.

Standard Kinetic Parameters Protocol:

• Prepare enzyme at known concentration (0.1-1.0 mg/mL in appropriate buffer)

• Set up reactions with varying substrate concentrations at optimal temperature

• Measure initial reaction velocities

• Calculate kinetic parameters (Km, kcat, Vmax) using appropriate models (Michaelis-Menten or Hill equation for cooperative enzymes)

Comparative Kinetic Parameters:

*Predicted based on ecological niche and related species

Allosteric Regulation Analysis:

• Sigmoidal velocity curves in response to increasing aspartate concentration indicate positive cooperativity

• Effects of nucleotides (ATP, CTP, UTP, GTP) on enzyme activity reveal allosteric regulatory mechanisms

• Comparison of holoenzyme vs. isolated catalytic trimer activity provides insights into the role of regulatory subunits

The kinetic analysis of Prochlorococcus marinus pyrB could reveal adaptations specific to its oligotrophic marine environment, potentially including modified substrate affinity or regulatory properties compared to model organisms.

What are the common challenges in expressing and purifying functional recombinant Prochlorococcus marinus pyrB, and how can they be addressed?

Researchers often encounter several challenges when working with recombinant Prochlorococcus proteins, including pyrB. Here are the most common issues and their solutions:

Expression Yield Challenges:

• Codon Bias:

  • Problem: Prochlorococcus has a relatively low G+C content (36.4% for P. marinus SS120) , which may result in codon usage incompatibility with expression hosts.

  • Solution: Use codon-optimized synthetic gene constructs or specialized expression strains with rare tRNA supplements (e.g., Rosetta DE3).

• Protein Solubility:

  • Problem: Recombinant pyrB may form inclusion bodies, particularly at high expression levels.

  • Solution: Reduce induction temperature to 16-20°C, decrease IPTG concentration (0.1-0.5 mM), or co-express with molecular chaperones (GroEL/ES, DnaK/J).

• Protein Stability:

  • Problem: Degradation during expression or purification.

  • Solution: Include protease inhibitors, express in protease-deficient hosts (BL21), and maintain samples at 4°C during purification .

Purification Challenges:

• Maintaining Native Structure:

  • Problem: Loss of oligomeric assembly or activity during purification.

  • Solution: Include carbamoyl phosphate (1-5 mM) in purification buffers to stabilize the catalytic trimer formation .

• Purity Concerns:

  • Problem: Contaminating proteins affect activity measurements.

  • Solution: Implement multi-step purification strategy combining affinity chromatography with ion exchange and size exclusion chromatography to achieve >85% purity .

Activity Assessment Challenges:

• Low Activity:

  • Problem: Recombinant enzyme shows reduced activity compared to native.

  • Solution: Verify proper folding using circular dichroism; ensure appropriate buffer conditions (pH, ionic strength) that mimic the marine environment.

• Reproducibility Issues:

  • Problem: Variable activity between preparations.

  • Solution: Standardize purification protocols; quantify protein using multiple methods (Bradford, BCA, A280); establish specific activity benchmarks.

Advanced Solution: Cell-Free Protein Synthesis

For particularly challenging proteins, cell-free protein synthesis systems offer an alternative approach:

• Prepare extract from E. coli or other suitable host

• Add template DNA (plasmid or PCR product)

• Supplement with amino acids, energy sources, and cofactors

• Incubate for 4-24 hours under controlled conditions

This method allows rapid testing of different conditions and bypasses issues related to cell toxicity or inclusion body formation.

How can structural biology techniques be integrated to fully characterize Prochlorococcus marinus pyrB structure-function relationships?

A comprehensive structural biology approach integrating multiple techniques provides the most complete understanding of pyrB structure-function relationships:

Integrative Structural Biology Workflow:

Functional Validation of Structural Features:

• Site-Directed Mutagenesis:

  • Target predicted catalytic residues

  • Assess effects on activity and substrate binding

  • Validate structural hypotheses

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Map solvent accessibility and dynamics

  • Identify conformational changes upon substrate binding

  • Complement static structural data with dynamic information

• Förster Resonance Energy Transfer (FRET):

  • Introduce fluorescent labels at specific positions

  • Measure distances between domains in solution

  • Monitor conformational changes in real-time

Data Integration Strategy:

Utilize integrative modeling platforms (e.g., Integrative Modeling Platform, IMP) to combine data from multiple experimental sources into a unified structural model, weighted by the resolution and reliability of each method.

This multi-technique approach would provide unprecedented insights into the structure-function relationships of Prochlorococcus marinus pyrB, potentially revealing adaptations specific to its marine environment.

What are the future research directions and unanswered questions regarding Prochlorococcus marinus pyrB?

Several intriguing research directions remain to be explored regarding Prochlorococcus marinus pyrB, offering opportunities for significant scientific contributions:

Evolutionary and Ecological Questions:

• Adaptive Evolution:

  • How has pyrB evolved across Prochlorococcus ecotypes adapted to different ocean depths and light regimes?

  • Are there signature mutations that correlate with specific environmental adaptations?

  • Do high- and low-light adapted strains show systematic differences in pyrB sequence or regulation?

• Horizontal Gene Transfer:

  • Is there evidence for horizontal gene transfer of pyrB or the entire pyrBI operon in Prochlorococcus?

  • The genome of Prochlorococcus contains a gene cluster for Rubisco and carboxysomal proteins that likely originated from non-cyanobacterial sources . Has pyrB undergone similar horizontal transfer events?

Structural and Functional Questions:

• Allosteric Regulation:

  • Does Prochlorococcus ATCase exhibit the same allosteric regulation as E. coli (inhibition by CTP, activation by ATP)?

  • How have regulatory mechanisms evolved in the context of genome streamlining?

• Quaternary Structure:

  • Does Prochlorococcus pyrB form the classical dodecameric structure (2(c3)·3(r2)) found in E. coli?

  • Are there structural adaptations that enhance function in the marine environment?

• Substrate Specificity:

  • Does Prochlorococcus ATCase show altered substrate specificity or catalytic efficiency compared to other bacterial homologs?

  • Could these differences reflect adaptation to the nutrient-limited marine environment?

Methodological Approaches to Address These Questions:

• Comparative Genomics and Evolution:

  • Sequence pyrB from diverse Prochlorococcus isolates across environmental gradients

  • Conduct selection analysis (dN/dS) to identify sites under positive selection

  • Reconstruct ancestral sequences to trace evolutionary trajectories

• Structure-Function Analysis:

  • Determine high-resolution structures using X-ray crystallography or cryo-EM

  • Perform enzyme kinetics across physiologically relevant conditions

  • Use site-directed mutagenesis to test hypotheses about catalytic mechanism

• Systems Biology Integration:

  • Analyze transcriptomic and proteomic data to understand pyrB regulation in environmental context

  • Develop metabolic models incorporating ATCase function in pyrimidine biosynthesis

  • Investigate interactions with other metabolic pathways and environmental response systems

Technical Innovations Needed:

• Improved methods for genetic manipulation of Prochlorococcus to enable in vivo studies

• Advanced structural biology approaches to capture conformational dynamics

• More sensitive analytical methods to measure enzyme activity at environmentally relevant scales

These research directions would not only enhance our understanding of Prochlorococcus marinus pyrB but could also provide broader insights into protein evolution, enzyme adaptation to extreme environments, and the molecular basis of ecological success in the world's oceans.