Recombinant Photobacterium profundum Fructose-1,6-bisphosphatase class 1 (fbp)

What is Fructose-1,6-bisphosphatase from Photobacterium profundum and what is its biochemical function?

Fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) from Photobacterium profundum is a key enzyme in gluconeogenesis that catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate. This reaction constitutes one of three reactions in gluconeogenesis that are not the exact reverse of corresponding reactions in glycolysis . In P. profundum, the FBPase enzyme is encoded by the fbp gene and plays a critical role in carbon metabolism under various environmental conditions, particularly in response to pressure changes. Unlike the glycolytic phosphofructokinase (PFK) which requires ATP to phosphorylate fructose 6-phosphate, FBPase-catalyzed dephosphorylation does not yield ATP, serving as a key regulatory point to prevent futile cycling between glycolysis and gluconeogenesis .

How does P. profundum FBPase differ from FBPases in other organisms?

P. profundum FBPase exhibits several distinct characteristics compared to FBPases from other organisms:

The P. profundum enzyme, particularly from strain SS9, shows adaptations that allow functionality under high-pressure deep-sea conditions, which is in stark contrast to mesophilic FBPases from terrestrial or shallow-water organisms . The computed structure model of P. profundum FBPase available in the AlphaFold DB (AF-Q6LV59-F1) shows high confidence scores, suggesting a well-defined structural arrangement that likely contributes to its pressure resistance .

What strain-specific variations exist in P. profundum FBPase?

Different strains of P. profundum show variations in FBPase that correlate with their native environmental conditions:

• Strain SS9: Isolated from the Sulu Trough at 2551m depth, this strain's FBPase is optimized for function at high pressure (28 MPa) and moderate temperature (15°C) .

• Strain 3TCK: Isolated from San Diego Bay, this shallow-water strain's FBPase is adapted to atmospheric pressure (0.1 MPa) and lower temperature (9°C) .

• Strain DSJ4: Obtained from the Ryukyu Trench at 5110m depth, its FBPase functions optimally at intermediate pressure (10 MPa) and low temperature (10°C) .

These strain-specific adaptations suggest evolutionary tuning of FBPase properties to match the specific ecological niches of each strain, making them valuable models for studying enzyme adaptation to extreme environments .

How can recombinant P. profundum FBPase be efficiently expressed and purified?

Expression and purification of recombinant P. profundum FBPase can be achieved through the following methodological approach:

• Vector selection: pET expression systems (such as pET22b+) are suitable for FBPase expression, as demonstrated in similar studies .

• Expression host: E. coli BL21(DE3) provides high expression levels while suppressing proteolytic degradation.

• Culture conditions:

  • Growth in M9 minimal medium with appropriate antibiotic

  • Induction at OD600 = 0.3 with 1 mM IPTG

  • Post-induction incubation at 26°C for 16 hours

• Purification strategy:

  • Periplasmic extraction using osmotic shock (30 mM Tris pH 8, 20% sucrose, 1 mM EDTA)

  • Ion exchange chromatography using a Q FF column with gradient elution (50 mM Tris pH 8 to 50 mM Tris pH 8, 1 M NaCl)

  • Size exclusion chromatography for final polishing

• Protein validation:

  • SDS-PAGE for purity assessment (>90% purity should be achieved)

  • Enzymatic activity assay measuring phosphate release or fructose 6-phosphate formation

The purified protein should be formulated in a stabilizing buffer (e.g., 20 mM Tris-HCl pH 8, 1 mM DTT, 10% glycerol) similar to that used for human FBPase, with storage at -20°C to maintain activity .

What experimental approaches can be used to characterize the pressure adaptation of P. profundum FBPase?

Several experimental approaches can be employed to characterize the pressure adaptation mechanisms of P. profundum FBPase:

• High-pressure enzyme kinetics:

  • Measure enzyme activity across a pressure range (0.1-70 MPa) using high-pressure vessels

  • Determine pressure-dependent changes in kinetic parameters (Km, Vmax, kcat)

  • Compare kinetic parameters of FBPase from different P. profundum strains (SS9, 3TCK)

• Structural analysis under pressure:

  • High-pressure X-ray crystallography to observe pressure-induced conformational changes

  • NMR spectroscopy under variable pressure conditions to monitor structural dynamics

  • Molecular dynamics simulations to predict pressure effects on enzyme flexibility

• Mutagenesis studies:

  • Site-directed mutagenesis of potential pressure-adaptive residues

  • Creation of chimeric enzymes between pressure-adapted (SS9) and pressure-sensitive (3TCK) FBPases

  • Functional analysis of mutants across pressure gradients

• Thermodynamic characterization:

  • Pressure-temperature phase diagrams for enzyme stability

  • Determination of activation volumes (ΔV‡) and reaction volumes (ΔV)

  • Measure pressure dependence of substrate and cofactor binding

These approaches have been successfully applied to other pressure-adapted enzymes from P. profundum and can be adapted specifically for FBPase characterization .

How does FBPase contribute to pressure adaptation in P. profundum SS9?

FBPase plays several critical roles in the pressure adaptation of P. profundum SS9:

• Metabolic rewiring: Under high pressure conditions, FBPase activity likely helps maintain appropriate gluconeogenic flux to compensate for pressure-induced changes in glycolysis. This metabolic adaptation ensures sufficient biosynthetic precursors under deep-sea conditions .

• Energy management: By controlling the direction of carbon flux between glycolysis and gluconeogenesis, FBPase contributes to pressure-optimized energy management strategies. This prevents futile cycling and conserves ATP, which is crucial under the energy-limited conditions of the deep sea .

• Gene expression patterns: Transcriptomic analyses reveal that FBPase gene expression in P. profundum SS9 responds to pressure changes, suggesting it participates in a coordinated gene expression response to hydrostatic pressure .

• Integration with stress response: FBPase regulation appears to be integrated with other pressure-responsive systems, including the ToxR regulon, which coordinates numerous pressure-adaptive genes in P. profundum SS9 .

The pressure adaptation of P. profundum FBPase thus represents not merely a single enzyme adaptation but part of a holistic cellular strategy for thriving under high hydrostatic pressure conditions.

What structural features of P. profundum FBPase contribute to its pressure resistance, and how can they be experimentally verified?

Several structural features likely contribute to the pressure resistance of P. profundum FBPase, with experimental verification strategies for each:

The high confidence score (pLDDT: 93.45) of the AlphaFold-predicted structure (AF-Q6LV59-F1) provides a valuable starting point for identifying these structural features . Comparing the P. profundum FBPase structure with that of non-pressure-adapted orthologs would reveal the specific structural adaptations that contribute to pressure resistance. These features could then be experimentally verified through the approaches outlined above, potentially informing the design of pressure-resistant enzymes for biotechnological applications.

How do the allosteric regulatory mechanisms of P. profundum FBPase compare to those in mammalian FBPases, and what are the implications for enzyme function under pressure?

Mammalian FBPases are subject to complex allosteric regulation, particularly through AMP and fructose 2,6-bisphosphate inhibition . This regulation involves information transmission between the AMP binding site and the active site, with specific residues like Lys112 and Tyr113 initiating the signal transduction . For P. profundum FBPase, the following comparative allosteric regulatory features and experimental approaches can be considered:

• Regulatory binding sites:

  • Computational prediction and structural analysis to identify potential allosteric sites

  • Isothermal titration calorimetry under variable pressure to measure binding affinities of potential allosteric effectors

  • Hydrogen-deuterium exchange mass spectrometry to map conformational changes upon effector binding

• Pressure effects on allostery:

  • Investigation of whether high pressure alters the binding of allosteric regulators

  • Testing if pressure changes the communication between allosteric and active sites

  • Characterization of pressure-dependent conformational equilibria using FRET-based sensors

• Signaling pathways:

  • Site-directed mutagenesis of putative signaling residues equivalent to Lys112 and Tyr113 in mammalian FBPases

  • Network analysis using molecular dynamics simulations to identify pressure-sensitive communication pathways

  • Comparison of pressure effects on wild-type versus allosteric mutants

The potentially unique allosteric mechanisms in P. profundum FBPase may represent evolutionary adaptations that enable fine-tuned metabolic control under high-pressure conditions, where conventional regulatory mechanisms might be compromised.

What role might P. profundum FBPase play in coordinating carbon metabolism with other pressure-responsive cellular processes?

P. profundum FBPase likely coordinates carbon metabolism with multiple pressure-responsive cellular processes, creating an integrated response to high-pressure environments:

• Integration with membrane adaptations:

  • FBPase activity may be coordinated with membrane lipid composition changes that occur under pressure

  • P. profundum SS9 modifies its fatty acid chains in response to pressure and temperature , and FBPase activity could provide metabolic precursors for these adaptations

  • Experimental approach: Lipidomic analysis combined with FBPase activity measurements across pressure gradients

• Coordination with stress response systems:

  • The pressure-stress response in P. profundum SS9 involves upregulation of genes like htpG, dnaK, dnaJ, and groEL

  • FBPase may be metabolically coupled to these stress response mechanisms

  • Experimental approach: Transcriptomic and proteomic analyses of wild-type versus fbp mutants under pressure stress

• Connection with motility systems:

  • P. profundum SS9 possesses two distinct flagellar systems adapted for high-pressure conditions

  • FBPase may contribute to energy allocation decisions between motility and other cellular functions

  • Experimental approach: Physiological characterization of swimming behavior in fbp mutants across pressure gradients

• Involvement in ToxR regulon:

  • The ToxR regulon coordinates numerous pressure-responsive genes in P. profundum

  • FBPase expression may be directly or indirectly regulated by this system

  • Experimental approach: Chromatin immunoprecipitation sequencing (ChIP-seq) to identify potential ToxR binding sites in the fbp promoter region

This multi-faceted coordination would allow P. profundum to maintain metabolic homeostasis while adapting to the challenges of its high-pressure habitat.

What insights can comparative studies of FBPases from different P. profundum strains provide about enzyme evolution under extreme conditions?

Comparative studies of FBPases from different P. profundum strains (SS9, 3TCK, DSJ4) provide valuable insights into enzyme evolution under extreme conditions:

• Evolutionary trajectory mapping:

  • Phylogenetic analysis of FBPase sequences from strains adapted to different depths

  • Identification of positively selected amino acid residues that correlate with pressure adaptation

  • Reconstruction of ancestral sequences to understand the stepwise evolution of pressure adaptation

• Structure-function relationship elucidation:

  • Comparative structural analysis of FBPases from pressure-adapted (SS9) and pressure-sensitive (3TCK) strains

  • Correlation of structural differences with functional parameters across pressure gradients

  • Creation and testing of chimeric enzymes to identify pressure-adaptive structural elements

• Horizontal gene transfer assessment:

  • Analysis of codon usage bias and GC content to identify potential horizontal gene transfer events

  • Comparison with FBPases from other deep-sea bacteria to detect convergent evolution

  • Investigation of whether FBPase adapts through gradual mutations or gene acquisition

• Ecological context integration:

  • Correlation of FBPase properties with the specific ecological niches of each strain

  • Analysis of how different selective pressures (temperature, pressure, nutritional status) shape FBPase evolution

  • In vitro evolution experiments mimicking ecological transitions to recapitulate natural evolutionary processes

These comparative approaches can reveal general principles of enzyme adaptation to extreme environments, potentially informing protein engineering strategies for creating pressure-resistant biocatalysts for various applications.