Recombinant Photobacterium profundum Aspartate carbamoyltransferase (pyrB)

What is Photobacterium profundum Aspartate carbamoyltransferase and what is its role in cellular metabolism?

Aspartate carbamoyltransferase (ATCase, encoded by the pyrB gene) is a key enzyme in pyrimidine nucleotide biosynthesis that catalyzes the reaction between L-aspartate and carbamoyl phosphate to form N-carbamoyl-L-aspartate and inorganic phosphate. In Photobacterium profundum, a deep-sea piezophilic bacterium, this enzyme has evolved to function optimally under high-pressure conditions (28 MPa) and low temperatures (15°C) .

The enzyme plays a critical role in regulating the pyrimidine biosynthetic pathway through allosteric mechanisms. Similar to the well-characterized E. coli ATCase, the P. profundum enzyme likely exhibits feedback inhibition by CTP and activation by ATP, allowing the bacterium to modulate nucleotide synthesis in response to cellular demands . Research with other bacterial species has shown that the pyrB gene is essential for survival, as demonstrated in studies with Helicobacter pylori where attempts to create pyrB mutants were unsuccessful .

How does the structure of P. profundum ATCase compare to other bacterial ATCases?

P. profundum ATCase shares structural similarities with other bacterial ATCases, particularly those from the Vibrionaceae family. Like E. coli ATCase (the most thoroughly characterized bacterial ATCase), the P. profundum enzyme is likely composed of catalytic subunits (encoded by pyrB) and regulatory subunits (encoded by pyrI) .

The quaternary structure of P. profundum ATCase probably resembles the classical arrangement found in E. coli ATCase, which consists of:

• Catalytic trimers (c3): Three pyrB-encoded chains that form the catalytic core

• Regulatory dimers (r2): Two pyrI-encoded chains that regulate enzyme activity

• Holoenzyme: An arrangement of two catalytic trimers and three regulatory dimers (c6r6)

Analysis of the amino acid sequence of P. profundum pyrB shows homology with other bacterial ATCases, containing the conserved domains necessary for substrate binding and catalysis, including residues involved in binding carbamoyl phosphate and aspartate .

What are the recommended methods for expressing recombinant P. profundum ATCase in heterologous systems?

Successfully expressing recombinant P. profundum ATCase requires considering both the host system and the unique adaptations of this deep-sea enzyme. Based on published methodologies for similar proteins, the following approaches are recommended:

Expression Systems:

Methodology:

• Clone the complete pyrB gene or pyrBI operon into an appropriate expression vector

• Transform into the selected host strain

• Induce expression at lower temperatures (15-20°C) to mimic native conditions

• For E. coli, use reduced IPTG concentrations (0.1-0.5 mM) and extend expression time (16-24 hours)

• For optimal activity, co-express both pyrB and pyrI genes to obtain the holoenzyme

What purification strategies yield the highest activity for recombinant P. profundum ATCase?

Purifying active P. profundum ATCase requires careful consideration of buffer conditions to maintain the enzyme's native conformation and activity. The following purification strategy is recommended based on successful approaches with related bacterial ATCases:

Purification Protocol:

• Cell Lysis: Use gentle lysis methods in a buffer containing 50 mM Tris-HCl (pH 7.5-8.0), 100-200 mM NaCl, 5% glycerol, and protease inhibitors. For pressure-adapted enzymes, avoiding high-pressure homogenization may better preserve native structure .

• Initial Purification: Apply cleared lysate to affinity chromatography (if using His-tagged constructs) or ion exchange chromatography (typically DEAE or Q-Sepharose) .

• Intermediate Purification: Employ size exclusion chromatography using a buffer system that mimics physiological conditions for P. profundum (containing 100-200 mM NaCl and 5-10% glycerol) .

• Final Polishing: Consider hydroxyapatite chromatography, which has been particularly effective for purifying ATCases from other organisms .

Critical Considerations:

• Maintain low temperature (4-10°C) throughout purification

• Include stabilizing agents like glycerol (10-20%) in all buffers

• For highest activity, ensure the quaternary structure remains intact by avoiding harsh conditions that might dissociate regulatory and catalytic subunits

• Consider adding 1-5 mM DTT or β-mercaptoethanol to protect thiol groups

What assay methods are most effective for measuring P. profundum ATCase activity under high-pressure conditions?

Measuring ATCase activity under high-pressure conditions requires specialized equipment and adapted protocols. Based on published methodologies for pressure-adapted enzymes, the following approaches are recommended:

Colorimetric Assays Under Pressure:

• Modified Prescott-Jones Colorimetric Assay: This classical method can be adapted for high-pressure conditions by using pressure-resistant cuvettes or microplate wells. The reaction measures the formation of N-carbamoyl-L-aspartate, which can be detected after reaction with specific colorimetric reagents .

Protocol for High-Pressure Enzymatic Assay:

• Prepare reaction mixture containing buffer (typically 50 mM Tris-HCl, pH 8.0), varying concentrations of L-aspartate (1-20 mM), and a fixed concentration of carbamoyl phosphate (5 mM)

• Place reaction mixture in specialized pressure vessels equipped with optical windows or in pressure-resistant containers

• Initiate reaction by adding enzyme solution

• Apply desired pressure (0.1-90 MPa) using a high-pressure generator

• Incubate at 15°C (optimal temperature for P. profundum)

• Stop reaction at different time points by adding acid

• Measure color development using a spectrophotometer

Alternative Approach:
For laboratories without specialized high-pressure equipment, a comparative approach can be used by assaying the enzyme at atmospheric pressure after pre-incubation at different pressures (0.1-90 MPa) to assess pressure effects on enzyme stability and structure .

How do temperature and pressure affect the kinetic parameters of P. profundum ATCase?

P. profundum ATCase, being from a piezophilic and psychrophilic organism, displays unique kinetic behaviors under different temperature and pressure conditions:

Temperature Effects:

• Optimal activity temperature: Approximately 15°C, corresponding to the optimal growth temperature of P. profundum SS9

• Activity decreases significantly above 25°C due to the cold-adapted nature of the enzyme

• At temperatures below 4°C, activity decreases but remains detectable, unlike mesophilic ATCases

Pressure Effects:

• Highest catalytic efficiency (kcat/Km) observed at 28 MPa, corresponding to the isolation depth of P. profundum SS9

• At atmospheric pressure (0.1 MPa), the enzyme exhibits reduced but measurable activity

• Pressure affects primarily the binding affinity for aspartate (Km) rather than the maximum reaction rate (Vmax)

• The cooperativity for aspartate binding (Hill coefficient) is likely pressure-dependent

Comparative Kinetic Parameters at Different Pressures:

These parameters reflect the evolutionary adaptation of P. profundum ATCase to function optimally under deep-sea conditions while maintaining sufficient activity across a range of pressures .

How does P. profundum ATCase achieve pressure adaptation at the molecular level?

The pressure adaptation of P. profundum ATCase involves several structural features that allow the enzyme to maintain flexibility and proper conformational changes under high hydrostatic pressure:

Molecular Adaptations for Pressure Resistance:

• Amino Acid Composition: Compared to ATCases from mesophilic organisms, P. profundum ATCase likely contains:

  • Reduced number of salt bridges that might be disrupted by pressure

  • Increased proportion of flexible amino acids (glycine, serine)

  • Reduced hydrophobic core packing to prevent pressure-induced rigidification

  • Strategic placement of charged residues to maintain critical electrostatic interactions under pressure

• Quaternary Structure Stability: The interfaces between catalytic and regulatory subunits are likely optimized to resist pressure-induced dissociation through:

  • Increased hydrogen bonding at subunit interfaces

  • Modified hydrophobic interactions at domain interfaces

  • Pressure-resistant allosteric communication pathways

• Active Site Architecture: The active site likely features:

  • More accessible binding pocket for substrates under pressure

  • Reduced volume change during catalytic transitions to minimize pressure sensitivity

  • Modified binding site for aspartate to maintain affinity at high pressure

• Conformational Flexibility: The T→R allosteric transition (crucial for ATCase function) is likely modified to occur efficiently under pressure through altered domain movement mechanics and modified tertiary structure elements .

These adaptations collectively enable P. profundum ATCase to maintain catalytic efficiency and regulatory properties under the high-pressure conditions of the deep sea .

How do the regulatory mechanisms of P. profundum ATCase compare to those of E. coli and other well-characterized ATCases?

P. profundum ATCase likely shares core regulatory mechanisms with E. coli ATCase but displays adaptations specific to deep-sea environments:

Comparative Regulatory Features:

• Allosteric Regulation:

  • Like E. coli ATCase, P. profundum ATCase is likely inhibited by CTP and activated by ATP

  • The magnitude of nucleotide effects is probably pressure-dependent, with stronger responses at higher pressures

  • The binding sites for allosteric effectors may have evolved different affinities optimized for high-pressure environments

• Cooperativity:

  • Exhibits positive cooperativity for aspartate binding, similar to E. coli ATCase

  • The degree of cooperativity (Hill coefficient) is likely pressure-modulated

  • The T→R conformational change critical for cooperativity may involve different energy barriers compared to mesophilic ATCases

• Substrate Binding:

  • The mechanism for substrate binding likely follows the ordered binding model (carbamoyl phosphate binds first, followed by aspartate)

  • The domain closure movement between the carbamoyl phosphate and aspartate domains may be modified to function efficiently under pressure

  • Key residues like Arg105, His134, and Thr55 (numbered according to E. coli sequence) are likely conserved but may have altered positioning

• Catalytic Mechanism:

  • The basic catalytic mechanism involving formation of a tetrahedral intermediate is likely conserved

  • The transition state stabilization may involve additional or modified interactions compared to E. coli ATCase to function under pressure

These regulatory adaptations allow P. profundum ATCase to respond appropriately to cellular metabolic needs in the high-pressure, low-temperature environment of the deep sea .

How can P. profundum ATCase be used as a model for studying pressure adaptation in enzymes?

P. profundum ATCase serves as an excellent model system for investigating fundamental principles of enzyme adaptation to extreme pressure environments:

Research Applications:

• Comparative Structural Biology:

  • Comparing the structures of P. profundum ATCase with mesophilic homologs (e.g., E. coli ATCase) can reveal specific adaptations to high pressure

  • Site-directed mutagenesis experiments can identify key residues responsible for pressure adaptation

  • Hybrid enzymes containing domains from piezophilic and mesophilic ATCases can help map pressure-adaptive regions

• Protein Engineering Studies:

  • The pressure-adaptive features of P. profundum ATCase can guide the engineering of pressure-resistant variants of industrial enzymes

  • Structure-based design approaches can incorporate identified pressure-adaptive motifs into other enzymes

  • Directed evolution experiments using P. profundum ATCase as starting material can generate enzymes with enhanced pressure resistance

• Biophysical Research:

  • Studies on the pressure-dependence of P. profundum ATCase can elucidate fundamental principles of protein volume changes during catalysis

  • High-pressure spectroscopic techniques (fluorescence, FTIR, NMR) applied to P. profundum ATCase can reveal conformational changes under pressure

  • Molecular dynamics simulations comparing piezophilic and mesophilic ATCases can provide atomic-level insights into pressure adaptation mechanisms

• Evolutionary Biochemistry:

  • Phylogenetic analysis of ATCases from various pressure environments can trace the evolutionary pathway to piezophilic adaptation

  • Ancestral sequence reconstruction and resurrection can identify key evolutionary transitions in pressure adaptation

What techniques can be used to study the conformational dynamics of P. profundum ATCase under varying pressure conditions?

Investigating the conformational dynamics of P. profundum ATCase under pressure requires specialized techniques that can capture structural information under non-ambient conditions:

Advanced Methodologies:

• High-Pressure X-ray Crystallography:

  • Diamond anvil cells allow X-ray diffraction data collection at pressures up to 100 MPa

  • Structural comparison of the T and R states under various pressures can reveal pressure effects on the allosteric transition

  • Time-resolved crystallography can potentially capture intermediate conformations during the T→R transition

• High-Pressure Spectroscopic Techniques:

  • FRET Analysis: Strategic placement of fluorophore pairs can monitor domain movements under pressure

  • High-Pressure NMR: Specialized high-pressure NMR cells can monitor chemical shift changes indicating conformational alterations

  • High-Pressure SAXS: Small-angle X-ray scattering under pressure can reveal quaternary structure changes

  • High-Pressure FTIR: Can monitor secondary structure changes under varying pressure conditions

• Single-Molecule Studies:

  • High-pressure microscopy chambers coupled with single-molecule FRET can observe individual enzyme molecules transitioning between conformational states

  • Optical tweezers or atomic force microscopy with pressure cells can measure mechanical properties and force-extension relationships under pressure

• Computational Approaches:

  • Molecular dynamics simulations at various pressures can predict conformational changes and energetic barriers

  • QM/MM methods can model the active site environment under pressure

  • Normal mode analysis can identify collective motions affected by pressure

  • Free energy calculations can quantify the pressure effect on the T→R equilibrium

Experimental Protocol for High-Pressure FRET Analysis:

• Introduce cysteine residues at strategic positions in the P. profundum ATCase sequence

• Label with appropriate FRET donor-acceptor pairs

• Place labeled enzyme in high-pressure optical cell

• Measure FRET efficiency at various pressures (0.1-90 MPa)

• Correlate FRET changes with enzyme activity measurements

• Calculate distance changes and construct pressure-dependent conformational models

What are common difficulties encountered when working with recombinant P. profundum ATCase and how can they be addressed?

Working with recombinant P. profundum ATCase presents several challenges due to its origin from a piezophilic, psychrophilic organism:

Expression Challenges:

Purification Challenges:

Activity Assay Challenges:

How should experimental conditions be optimized when studying the effect of pressure on P. profundum ATCase activity?

Optimizing experimental conditions for studying pressure effects on P. profundum ATCase requires careful consideration of multiple parameters:

Buffer Optimization:

• pH Considerations:

  • Test pH range 7.0-8.5 at different pressures (pH can shift under pressure)

  • Use pressure-stable buffers like HEPES or PIPES

  • Measure and adjust pH at the temperature used for assays (15°C)

• Salt Effects:

  • Optimize NaCl concentration (typically 100-300 mM)

  • Test different cations (K+, NH4+) as they may differently affect pressure responses

  • Include divalent cations (Mg2+, Mn2+) at varying concentrations to stabilize the enzyme

Control Experiments:

• Thermal Stability Controls:

  • Perform thermal denaturation studies at different pressures

  • Establish pressure-temperature stability map

  • Pre-incubate enzyme at various pressure-temperature combinations before activity testing

• Comparative Controls:

  • Include E. coli ATCase as a mesophilic control

  • If available, test ATCase from related Photobacterium species adapted to different depths

  • Use catalytic subunits alone (without regulatory subunits) to isolate pressure effects on catalysis versus regulation

Pressure Application Protocol:

• Pressure Range and Steps:

  • Test fine increments around the optimal pressure (20-35 MPa)

  • Include atmospheric pressure (0.1 MPa) as baseline

  • Include extreme pressure points (60-90 MPa) to establish full pressure-response curve

• Pressure Application Rate:

  • Apply pressure gradually (5-10 MPa/min) to prevent denaturation

  • Allow equilibration time (5-10 min) at each pressure point

  • Consider pressure cycling experiments to test reversibility

• Data Analysis:

  • Plot data using various models (Michaelis-Menten, Hill equation)

  • Analyze pressure effects on individual kinetic parameters (Km, Vmax, Hill coefficient)

  • Apply transition state theory models to extract activation volume (ΔV‡) information

Example Experimental Matrix:

This systematic approach will generate a comprehensive dataset revealing how different environmental factors interact with pressure to affect enzyme function .

What are the current knowledge gaps regarding P. profundum ATCase structure and function?

Despite the importance of P. profundum ATCase as a model for pressure adaptation, several key knowledge gaps remain:

Structural Gaps:

• High-Resolution Structure: No crystal or cryo-EM structure of P. profundum ATCase currently exists, limiting our understanding of its precise structural adaptations to pressure .

• Conformational States: The T and R states of P. profundum ATCase have not been characterized structurally, preventing detailed understanding of how the allosteric transition occurs under pressure .

• Pressure Effects on Quaternary Structure: The stability of the holoenzyme complex under varying pressure conditions remains poorly characterized .

Functional Gaps:

• Pressure-Dependent Allostery: The precise mechanism by which pressure modulates the response to allosteric effectors (ATP, CTP) remains unclear .

• Catalytic Mechanism Under Pressure: How pressure affects the stabilization of the transition state during catalysis is not well understood .

• In vivo Regulation: How P. profundum regulates ATCase activity in vivo under native deep-sea conditions remains largely unexplored .

Evolutionary Gaps:

• Molecular Basis of Adaptation: The specific amino acid substitutions responsible for pressure adaptation have not been systematically identified .

• Evolutionary Pathway: The evolutionary trajectory from mesophilic to piezophilic ATCase remains unclear .

• Comparative Analysis: Comprehensive comparison with ATCases from related Photobacterium species adapted to different depths would provide evolutionary insights .

What innovative approaches could advance our understanding of pressure effects on enzyme catalysis using P. profundum ATCase as a model?

Advancing our understanding of P. profundum ATCase requires innovative approaches that combine cutting-edge technologies with creative experimental designs:

Emerging Methodologies:

• Structural Biology Innovations:

  • Serial Crystallography Under Pressure: Using X-ray free-electron lasers (XFELs) with high-pressure sample environments to capture structural snapshots

  • Time-Resolved Cryo-EM: Capturing conformational transitions under pressure followed by rapid freezing

  • Neutron Crystallography: To locate hydrogen atoms and protonation states under pressure conditions

• Advanced Computational Approaches:

  • Machine Learning: Using AI to predict pressure effects on protein dynamics based on sequence information

  • Enhanced Sampling Methods: Applying techniques like metadynamics or replica exchange to overcome energy barriers in simulations

  • Quantum Mechanical Modeling: Applying quantum approaches to model electronic effects of pressure on catalysis

• Systems Biology Approaches:

  • Multi-omics Analysis: Combining proteomics, transcriptomics, and metabolomics to understand ATCase regulation in the context of the entire pyrimidine pathway under pressure

  • In vivo Activity Measurements: Developing techniques to measure ATCase activity within living P. profundum cells under pressure

  • Synthetic Biology: Engineering minimal systems containing only essential components of the pyrimidine pathway to study pathway regulation under pressure

• Evolutionary Biochemistry:

  • Ancestral Sequence Reconstruction: Resurrecting ancestral ATCases to trace the evolutionary pathway to pressure adaptation

  • Horizontal Gene Transfer Analysis: Investigating if pressure adaptation features were acquired through horizontal gene transfer

  • Deep Mutational Scanning: Systematically analyzing thousands of ATCase variants to map the fitness landscape under pressure

• Interdisciplinary Approaches:

  • Biophysical Ecology: Correlating ATCase adaptations with ecological distribution of Photobacterium species across depth gradients

  • Astrobiology Applications: Using insights from pressure adaptation to understand potential enzyme function in high-pressure extraterrestrial environments (e.g., Europa's subsurface ocean)

  • Biomimetic Materials: Applying principles from pressure-adapted enzymes to design pressure-resistant synthetic catalysts

Future Research Directions:

• Development of a comprehensive pressure-temperature-pH phase diagram for P. profundum ATCase activity

• Comparative analysis of ATCases from the complete pressure spectrum (shallow water to hadal depths)

• Creation of chimeric enzymes combining domains from piezophilic and mesophilic ATCases to map pressure-adaptive features

• Investigation of co-evolution between ATCase and other enzymes in the pyrimidine pathway under pressure adaptation

• Application of pressure-adaptive principles from P. profundum ATCase to engineer pressure-resistant biocatalysts for industrial applications