Recombinant Photobacterium profundum Isocitrate dehydrogenase kinase/phosphatase (aceK), partial

What is the physiological role of AceK in Photobacterium profundum?

AceK in P. profundum likely serves as a bifunctional enzyme that regulates isocitrate dehydrogenase (ICDH) activity through reversible phosphorylation, similar to its homolog in E. coli. This regulation allows the bacterium to adjust its metabolic pathways in response to environmental changes, particularly hydrostatic pressure. In E. coli, AceK phosphorylates or dephosphorylates ICDH, resulting in the inactivation or activation of ICDH respectively, which enables the switching between the Krebs cycle and the glyoxylate bypass . In the context of P. profundum, this regulation may be particularly important for adaptation to deep-sea environments, as different strains (such as the piezophilic SS9 and the non-piezophilic 3TCK) display remarkable differences in their physiological responses to pressure . The genome analyses of these strains reveal variations in gene content and specific gene sequences under positive selection, which likely contribute to their differential adaptation to depth in the water column.

How does P. profundum AceK differ from E. coli AceK in terms of structure and function?

While detailed structural information specific to P. profundum AceK is limited in the literature, comparative analysis with E. coli AceK provides valuable insights. E. coli AceK is a 65-kDa protein with a eukaryotic protein-kinase-like domain containing ATP and a regulatory domain with a novel fold . It has both kinase and phosphatase activities within one protein and specifically recognizes only intact ICDH . P. profundum AceK likely shares these fundamental characteristics but may possess adaptations that enable function under high hydrostatic pressure conditions. These adaptations could include modifications in the active site architecture, substrate binding affinity, or allosteric regulation mechanisms. In particular, P. profundum strain SS9, isolated from deep-sea environments, shows adaptations to high hydrostatic pressure that are not present in shallow-water strains like 3TCK . These adaptations may extend to the structure and function of AceK, potentially resulting in pressure-optimized enzymatic activities.

What genomic features characterize the aceK gene in different P. profundum strains?

The aceK gene in P. profundum likely exhibits strain-specific variations that correlate with adaptation to different hydrostatic pressure environments. The genome of P. profundum strain 3TCK (6,186,725 bp with 41.3% GC content) encodes for 5549 ORFs and is organized in two chromosomes, similar to other members of the Vibrionaceae family . Although specific details about the aceK gene were not directly mentioned in the search results, the genomic analysis reveals that P. profundum strains differ in their large intergenic regions, with the deep-sea piezopsychrophilic strain SS9 having larger intergenic regions (~205 bp) compared to the shallow-water strain 3TCK (~167 bp) . These large intergenic regions have been shown to be transcribed and differentially expressed as a function of pressure, suggesting they could play an important physiological role . The aceK gene might be subject to similar strain-specific variations that define the Hutchinsonian niche of each strain, ranging from variations in gene content to specific gene sequences under positive selection.

What are the optimal methods for cloning and expressing recombinant P. profundum AceK?

For successful cloning and expression of recombinant P. profundum AceK, researchers should consider the following methodological approach:

• Gene Amplification: Amplify the aceK gene using PCR with high-fidelity DNA polymerase. Design primers based on the known genome sequence of P. profundum (e.g., strain SS9 or 3TCK). A typical PCR protocol would include cycles of 92°C for 1 min, 48-50°C for 1 min, and 72°C for 1-1.5 min for 25 cycles, similar to that used for amplifying other P. profundum genes .

• Cloning Vector Selection: Clone the amplified gene into an appropriate vector system. For initial cloning, vectors like pCR2.1 can be used, followed by subcloning into expression vectors compatible with the host system . For expression in E. coli, vectors like pGL10 or pUC18 have been successfully used with P. profundum genes .

• Conjugal Transfer: If working with P. profundum as the expression host, employ tri-parental conjugations using a helper E. coli strain (e.g., pRK2073) as described for other P. profundum genes. Matings can be performed at room temperature for 12-16 hours, with exconjugants selected on appropriate media and incubated at 15°C for 3-5 days .

• Expression Conditions: When expressing in E. coli, consider temperature optimization (likely lower than standard 37°C), as P. profundum is a psychrophilic organism. For expression in P. profundum, hydrostatic pressure conditions should be optimized based on the strain used (e.g., 280 atm for strain SS9) .

• Protein Purification: Include a fusion tag (His-tag or GST) to facilitate purification, and employ chromatographic techniques appropriate for the fusion system used.

What challenges exist in purifying active P. profundum AceK and how can they be addressed?

Purifying active P. profundum AceK presents several challenges that researchers must address:

• Pressure Adaptation: P. profundum AceK, especially from piezophilic strains like SS9, may be adapted to function optimally under high pressure. Standard purification protocols performed at atmospheric pressure might result in conformationally altered or inactive enzyme. Solution: Develop specialized high-pressure equipment for protein purification or rapidly purify the enzyme at atmospheric pressure while maintaining low temperature to minimize structural alterations.

• Temperature Sensitivity: As P. profundum is a psychrophilic organism, its enzymes including AceK may be heat-labile. Solution: Perform all purification steps at low temperatures (4-10°C) and minimize exposure to room temperature.

• Bifunctional Activity Preservation: Maintaining both kinase and phosphatase activities during purification can be challenging. Solution: Carefully optimize buffer conditions, including pH, salt concentration, and presence of stabilizing agents like glycerol or specific ions that may be required for structural integrity.

• Substrate Availability: For activity assays, ensuring access to properly folded ICDH substrate is crucial, as E. coli AceK specifically recognizes only intact ICDH . Solution: Co-express or separately purify P. profundum ICDH for use in activity assays.

• Pressure-Dependent Activity Assessment: Standard enzyme assays are typically performed at atmospheric pressure. Solution: Develop specialized high-pressure enzyme assay systems to characterize the pressure-dependent activity profile of the enzyme.

How can researchers effectively measure the dual kinase and phosphatase activities of P. profundum AceK?

To effectively measure the dual kinase and phosphatase activities of P. profundum AceK, researchers should employ a comprehensive approach:

Table 1: Methodological Approaches for Measuring AceK Dual Activities

The kinase activity can be measured by monitoring the decrease in ICDH activity as it becomes phosphorylated, using a spectrophotometric assay that follows NADPH production. The phosphatase activity can be assessed by measuring the restoration of ICDH activity from previously phosphorylated ICDH. Additionally, AceK's intrinsic ATPase activity should be measured as a control.

For accurate characterization of a piezophilic enzyme, these assays should ideally be performed at different hydrostatic pressures. Since E. coli AceK activities are regulated by various metabolites including AMP (which acts as a phosphatase activator and kinase inhibitor) , researchers should investigate how these allosteric regulations are affected by pressure in P. profundum AceK.

What structural features of P. profundum AceK might contribute to pressure adaptation?

Several structural features of P. profundum AceK may contribute to its adaptation to high hydrostatic pressure, particularly in piezophilic strains like SS9:

• Increased Flexibility in Active Site Regions: Pressure-adapted enzymes often show increased flexibility in active site regions to maintain catalytic efficiency under compression. In P. profundum AceK, the kinase and phosphatase active sites might have evolved specific amino acid substitutions that confer greater conformational flexibility under high pressure.

• Modified ATP Binding Pocket: The ATP binding motif, which is conserved between AceK and typical eukaryotic protein kinases , may exhibit pressure-specific adaptations in P. profundum. These adaptations could include changes in hydrophobicity or charge distribution to maintain optimal ATP binding under pressure.

• Altered Allosteric Regulation: In E. coli AceK, AMP binds in an allosteric site between the two AceK domains and mediates a conformational change that switches between kinase and phosphatase activities . P. profundum AceK may have evolved pressure-sensitive allosteric sites that respond differently to regulatory metabolites under varying hydrostatic pressures.

• Pressure-Optimized Domain Interface: The interface between the kinase-like domain and the regulatory domain of AceK is crucial for its function. In P. profundum, this interface may have evolved to maintain optimal interdomain communication under pressure.

• Modified ICDH Recognition Elements: The substrate recognition loop of AceK binds to the ICDH dimer . In P. profundum, this recognition element may be adapted to maintain proper substrate interaction under high pressure.

These structural adaptations would be consistent with the genomic plasticity observed between P. profundum strains from different depths, which show variations in gene content and specific gene sequences under positive selection .

How does hydrostatic pressure affect the catalytic efficiency of P. profundum AceK?

Hydrostatic pressure likely exerts complex effects on the catalytic efficiency of P. profundum AceK, with distinct patterns expected for deep-sea piezophilic strains (like SS9) versus shallow-water strains (like 3TCK):

• Rate-Limiting Step Modulation: High pressure typically affects the rate-limiting step of enzymatic reactions by influencing the activation volume. For deep-sea P. profundum AceK, the enzyme may have evolved a smaller activation volume for catalysis, minimizing the inhibitory effects of pressure on reaction rates.

• Substrate Binding Kinetics: Pressure affects association-dissociation equilibria based on volume changes. The ICDH-binding interface of AceK from piezophilic strains may have evolved to maintain optimal substrate affinity under high pressure, possibly through modifications in the substrate recognition loop that binds to the ICDH dimer .

• Allosteric Regulation Sensitivity: The pressure sensitivity of AceK's bifunctionality switch may differ between strains. In E. coli, AMP acts as a phosphatase activator and kinase inhibitor by binding in an allosteric site between AceK domains . In P. profundum, this regulatory mechanism may be tuned to respond optimally to metabolite levels at specific pressure ranges corresponding to the strain's native depth.

• ATP Utilization Efficiency: Given that AceK possesses strong ATPase activity , pressure may differently affect ATP hydrolysis versus phosphotransfer activities. Piezophilic P. profundum AceK may have evolved to maintain a favorable ratio of these activities under high pressure.

• Conformational Stability: Pressure-induced conformational changes could affect the exposure of catalytic residues. Piezophilic P. profundum AceK likely maintains optimal conformational states under high pressure, possibly through adaptations similar to those observed in other pressure-adapted proteins from strain SS9.

Understanding these effects requires comparative enzymatic studies of AceK from different P. profundum strains under varying pressure conditions, similar to approaches used for other pressure-adapted enzymes in this organism.

What insights can computational modeling provide about P. profundum AceK structure under varying pressure conditions?

Computational modeling offers valuable insights into P. profundum AceK structure under varying pressure conditions, addressing challenges in experimental high-pressure structural biology:

• Homology Modeling: Using the E. coli AceK structure as a template, researchers can generate baseline models of P. profundum AceK. By comparing predicted structures from piezophilic (SS9) and non-piezophilic (3TCK) strains, key structural differences that might contribute to pressure adaptation can be identified, focusing on:

  • The kinase-like domain containing the ATP binding site

  • The regulatory domain with its novel fold

  • The allosteric site where AMP binds between domains

  • The substrate recognition loop that interacts with ICDH

• Molecular Dynamics Simulations: Pressure-explicit MD simulations can reveal how different P. profundum AceK variants respond to pressure changes:

  • Conformational flexibility analysis under different pressures (1-1000 atm)

  • Water penetration into protein cavities, which often occurs under pressure

  • Changes in hydrogen bonding networks and salt bridges

  • Domain movement dynamics, particularly how pressure affects the conformational switch between kinase and phosphatase states

• Binding Energy Calculations: Computational methods can predict how pressure affects:

  • ATP binding affinity and positioning

  • ICDH substrate recognition and binding

  • AMP and other allosteric regulators' interactions

  • Pressure-dependent changes in these energetics

• Quantum Mechanics/Molecular Mechanics (QM/MM): For detailed understanding of the catalytic mechanism under pressure:

  • Calculation of activation barriers for phosphorylation/dephosphorylation reactions

  • Identification of pressure-sensitive steps in the catalytic mechanism

  • Prediction of catalytic efficiency changes with pressure

• Evolutionary Analysis and Rational Design: By integrating computational predictions with experimental data:

  • Identification of key residues for pressure adaptation through evolutionary analysis

  • Rational design of mutations to enhance or alter pressure-dependent activity

  • Prediction of functional consequences of naturally occurring variants in different P. profundum strains

These computational approaches complement experimental studies and help formulate testable hypotheses about structure-function relationships in P. profundum AceK under varying pressure conditions.

How does P. profundum AceK compare to AceK from other marine bacteria in terms of pressure adaptation?

P. profundum AceK likely exhibits unique pressure adaptations compared to AceK from other marine bacteria, reflecting the organism's evolutionary history and ecological niche:

• Strain-Specific Adaptations: Different P. profundum strains show remarkable differences in their pressure responses, with deep-sea strains (SS9, DSJ4) adapted to high hydrostatic pressure and shallow-water strains (3TCK, 1230sf1) inhibited by elevated pressure . This suggests that AceK from these strains may exhibit corresponding differences in pressure optima for enzymatic activity, with SS9 AceK likely showing enhanced function at high pressure (280 atm).

• Comparative Genomic Context: The genomic context of aceK may differ between marine bacteria. In P. profundum, larger-than-average intergenic regions (approximately 167-205 bp) are present and transcribed, with differential expression as a function of pressure . These genomic features may influence aceK expression regulation in response to pressure changes.

• Evolutionary Rate and Selection: Pressure adaptation in P. profundum appears to evolve rapidly, as suggested by the existence of phylogenetically cohesive bathytypes that differ in their adaptation to depth . This rapid evolution may extend to AceK, potentially resulting in lineage-specific amino acid substitutions that optimize function under particular pressure conditions.

• Functional Adaptation Mechanisms: While E. coli AceK functions as a bifunctional enzyme with the kinase, phosphatase, and ATPase activities residing at the same site , marine bacterial AceK enzymes may have evolved different mechanisms for regulating these activities under pressure. P. profundum AceK might show pressure-dependent shifts in the balance between kinase and phosphatase activities that are not observed in non-piezophilic marine bacteria.

• Metabolic Integration: The role of AceK in regulating the switch between the Krebs cycle and glyoxylate bypass may take on additional significance in pressure-adapted organisms. P. profundum SS9 exhibits various physiological responses to pressure, including upregulation of chaperones and DNA repair enzymes under suboptimal pressure , suggesting that metabolic regulation by AceK may be integrated with these broader pressure response networks.

Detailed comparative biochemical studies are needed to fully characterize these differences, similar to approaches used for analyzing other pressure-adapted enzymes from P. profundum.

What methodological approaches reveal differences between E. coli and P. profundum AceK structure-function relationships?

To systematically investigate differences between E. coli and P. profundum AceK structure-function relationships, researchers should employ a multi-faceted methodological approach:

Table 2: Comparative Methodological Approaches for E. coli vs. P. profundum AceK Analysis

• Structural Analysis: Comparing crystal structures of E. coli and P. profundum AceK would reveal differences in active site architecture, regulatory domain organization, and allosteric sites. The E. coli AceK structure shows a eukaryotic protein-kinase-like domain containing ATP and a regulatory domain with a novel fold . P. profundum AceK structures should be obtained in both substrate-free and ICDH-bound states to identify strain-specific adaptations.

• Domain Swapping Experiments: Creating chimeric proteins with domains from E. coli and P. profundum AceK would help identify which regions confer pressure adaptation. This approach is similar to genetic complementation studies performed with P. profundum RecD, where the introduction of SS9 recD into an E. coli recD mutant enabled growth under high pressure .

• Allosteric Regulation Analysis: Since AMP acts as a switch between AceK kinase and phosphatase activities in E. coli through binding to an allosteric site , comparative analysis of allosteric regulation in P. profundum AceK under different pressure conditions would reveal adaptations in regulatory mechanisms.

• Substrate Recognition Studies: E. coli AceK specifically recognizes only intact ICDH dimers . Investigating whether P. profundum AceK exhibits pressure-dependent changes in substrate recognition specificity would provide insights into functional adaptations.

• Heterologous Expression Analysis: Following the approach used with RecD , expressing P. profundum AceK in E. coli and vice versa can reveal host-dependent activity patterns and pressure sensitivity.

These methodological approaches would systematically identify the structural and functional differences that distinguish E. coli and P. profundum AceK, particularly with respect to pressure adaptation.

How do evolutionary pressures in different marine environments shape AceK diversification?

Evolutionary pressures in different marine environments have likely shaped significant AceK diversification, particularly in relation to hydrostatic pressure adaptation:

• Depth-Dependent Selection: The water column represents a continuous gradient of hydrostatic pressure, temperature, and nutrient availability. These environmental factors exert strong selective pressures that have led to the evolution of distinct P. profundum bathytypes adapted to specific depth ranges . The genetic modifications required for depth-specific adaptations appear to evolve rapidly, suggesting that enzymes like AceK may show corresponding adaptations to function optimally at specific pressure ranges.

• Vertical Migration Influences: The existence of advective transport phenomena (up/downwelling, Ekman transport, thermohaline circulation) can result in bathytype conversion as microbial communities are moved between different depths . This vertical migration creates evolutionary pressure for metabolic flexibility, potentially driving the evolution of pressure-responsive regulatory mechanisms in AceK.

• Metabolic Adaptation Signatures: The regulation of the switch between the Krebs cycle and glyoxylate bypass by AceK is fundamental for adapting to changing carbon sources. In different marine environments, this regulation may be fine-tuned to match available carbon sources at different depths, leading to diversification in AceK regulatory properties.

• Genomic Context Evolution: P. profundum strains show notable differences in genomic features, including variations in gene content, specific gene sequences under positive selection, and intergenic region sizes . These genomic changes likely affect the evolution of AceK through altered gene expression regulation, protein-protein interactions, and metabolic network integration.

• Horizontal Gene Transfer Dynamics: Marine environments facilitate horizontal gene transfer, which could contribute to AceK diversification. The observation that introducing SS9 recD into an E. coli recD mutant enables growth under high pressure suggests that pressure-adapted genes can confer fitness advantages across species, potentially driving selection for or against gene transfer events involving aceK.

Understanding these evolutionary dynamics requires comparative genomic and biochemical analyses of AceK from multiple marine bacterial species across depth gradients, combined with experimental evolution studies under varying pressure conditions.

What are the primary technical challenges in studying pressure effects on P. profundum AceK activity?

Researchers face several significant technical challenges when studying pressure effects on P. profundum AceK activity:

• High-Pressure Experimental Equipment: Specialized equipment is required to study enzymatic activity under high hydrostatic pressure (up to 280 atm for deep-sea strain SS9) . These high-pressure vessels need to be compatible with spectrophotometric measurements for real-time activity monitoring, requiring transparent windows that can withstand pressure while allowing light transmission. The design and calibration of such equipment present significant engineering challenges.

• Real-Time Activity Measurement: Continuous monitoring of the dual kinase/phosphatase activities under pressure requires sophisticated approaches. While discontinuous assays (sampling at pressure followed by activity measurement at atmospheric pressure) are simpler, they may not capture transient pressure effects on enzyme conformation and activity.

• Protein Stability Under Pressure: Maintaining protein stability during high-pressure experiments is challenging. Proteins may undergo pressure-induced unfolding or aggregation, particularly for non-piezophilic variants. Developing stabilizing buffer conditions that do not interfere with activity measurements requires extensive optimization.

• AceK-ICDH Complex Formation: Since AceK specifically recognizes only intact ICDH dimers , studying the pressure dependence of this protein-protein interaction adds complexity. The pressure stability of both the enzyme and substrate must be considered, and methods for monitoring complex formation under pressure are technically demanding.

• Pressure Effects on Allosteric Regulation: The allosteric regulation of AceK by metabolites like AMP, which acts as a phosphatase activator and kinase inhibitor , may be pressure-dependent. Assessing these complex regulatory interactions under varying pressure conditions requires sophisticated experimental designs capable of detecting subtle changes in regulatory responses.

• Genetic Manipulation Under Pressure: Creating and characterizing AceK mutants requires genetic tools that work effectively with P. profundum under varying pressure conditions. While conjugal transfer methods have been developed for P. profundum , their efficiency may vary with different strains and pressure conditions, complicating genetic studies of AceK function.

Addressing these challenges requires interdisciplinary approaches combining protein biochemistry, biophysics, genetic engineering, and specialized instrumentation design.

How can researchers effectively study the role of AceK in P. profundum's adaptation to varying hydrostatic pressures?

To effectively study AceK's role in P. profundum's pressure adaptation, researchers should implement a comprehensive, multi-level research strategy:

• Comparative Genomics and Transcriptomics:

  • Analyze aceK sequence variations across P. profundum strains from different depths (SS9, DSJ4, 3TCK, 1230sf1)

  • Perform RNA-seq under varying pressure conditions to determine if aceK expression changes with pressure, similar to studies that have revealed differential expression of ATP synthases and membrane transporters in response to pressure

  • Use comparative genomics to identify potential regulatory elements in the larger-than-average intergenic regions that might influence aceK expression in response to pressure

• Genetic Manipulation:

  • Create aceK knockout mutants in both piezophilic (SS9) and non-piezophilic (3TCK) strains using established conjugation methods for P. profundum

  • Develop complementation systems with aceK variants from different strains

  • Generate site-directed mutants targeting predicted pressure-responsive regions

  • Assess growth phenotypes of these genetic variants under different pressure conditions

• Biochemical Characterization:

  • Purify recombinant AceK from different P. profundum strains

  • Measure kinase, phosphatase, and ATPase activities under varying pressure conditions (1-280 atm)

  • Determine pressure-dependent changes in substrate affinity, catalytic efficiency, and allosteric regulation

  • Assess the pressure stability of the AceK-ICDH complex

• Structural Analysis:

  • Obtain crystal structures or use cryo-EM to determine AceK structures from different P. profundum strains

  • Employ hydrogen-deuterium exchange mass spectrometry to map pressure-induced conformational changes

  • Use small-angle X-ray scattering under pressure to assess global structural changes

• Metabolic Network Integration:

  • Develop metabolic flux models for P. profundum under different pressure conditions

  • Determine how AceK-mediated regulation of the Krebs cycle/glyoxylate bypass switch responds to pressure

  • Integrate AceK function with other pressure-responsive elements of P. profundum metabolism

• Evolutionary Analysis:

  • Reconstruct the evolutionary history of aceK in marine bacteria across depth gradients

  • Identify positively selected residues that may contribute to pressure adaptation

  • Perform experimental evolution studies under varying pressure conditions to observe real-time adaptation

This multi-faceted approach will provide comprehensive insights into how AceK contributes to P. profundum's remarkable ability to adapt to varying hydrostatic pressures in marine environments.

What potential applications might emerge from understanding P. profundum AceK's pressure-adaptive mechanisms?

Understanding P. profundum AceK's pressure-adaptive mechanisms could lead to several significant scientific and biotechnological applications:

• Enzyme Engineering for Extreme Conditions:

  • Design of pressure-resistant enzymes for industrial biocatalysis under non-standard conditions

  • Development of enzymes with pressure-switchable activity states for controlled bioprocessing

  • Creation of biosensors that respond to pressure changes in deep-sea environments

  • Engineering metabolic pathways that function efficiently under high-pressure conditions

• Biomedical Applications:

  • Insights into protein function under mechanical stress, relevant to understanding cellular responses to pressure and mechanical forces in human tissues

  • Development of pressure-stable therapeutic proteins with extended shelf-life

  • Design of pressure-responsive drug delivery systems based on protein conformational changes

  • Understanding protein misfolding diseases through the lens of pressure-induced conformational changes

• Astrobiology and Exobiology:

  • Models for enzyme function in extraterrestrial environments with different pressure regimes (e.g., Europa's subsurface ocean)

  • Insights into the limits of protein-based life under extreme conditions

  • Development of biomarkers for detecting life in high-pressure extraterrestrial environments

• Deep-Sea Biotechnology:

  • Creation of microbial cell factories optimized for deep-sea bioprospecting and in situ bioremediation

  • Development of pressure-adapted biocatalysts for accessing and processing deep-sea resources

  • Design of pressure-stable enzymes for cold-adapted industrial processes that benefit from low-temperature, high-pressure conditions

• Fundamental Protein Science:

  • New principles of protein structure-function relationships under pressure

  • Understanding allosteric regulation mechanisms that respond to physical parameters beyond temperature and pH

  • Insights into protein evolution in response to physical stress parameters

  • Development of new theoretical frameworks for predicting pressure effects on protein function

• Climate Change Research:

  • Tools for monitoring metabolic adaptations of marine microorganisms to changing ocean conditions

  • Understanding how ocean circulation changes might affect microbial communities through pressure adaptation mechanisms

  • Predicting impacts of changing deep-sea conditions on biogeochemical cycles mediated by pressure-adapted organisms

These applications highlight the broader significance of understanding AceK's pressure adaptations beyond basic science, with potential impacts across multiple disciplines and technologies.