Recombinant Photobacterium profundum Anthranilate phosphoribosyltransferase (trpD)

What is Anthranilate phosphoribosyltransferase (TrpD) and what role does it play in P. profundum?

Anthranilate phosphoribosyltransferase (TrpD) is an enzyme that catalyzes the second step in tryptophan biosynthesis, transferring a phosphoribosyl group to anthranilate to generate phosphoribosyl anthranilate (PRA), which forms the basic skeleton of tryptophan . In Photobacterium profundum, a deep-sea bacterium, this enzyme is particularly important as it contributes to the organism's survival under high-pressure conditions. While the specific adaptations of P. profundum TrpD haven't been fully characterized, it likely possesses unique properties that enable it to function efficiently in the deep-sea environment where P. profundum naturally inhabits.

What is known about the genetic organization of trpD in P. profundum?

While the specific genomic context of the trpD gene in P. profundum is not fully detailed in the provided search results, we can infer some information based on knowledge of related bacteria. P. profundum has a multi-chromosome genome structure, with chromosome II replication being regulated by the RctB protein . The trpD gene is likely part of the tryptophan operon, which typically includes genes encoding other enzymes in the tryptophan biosynthesis pathway. The organization of this operon and its regulation mechanisms may have specific adaptations related to the deep-sea environment where P. profundum thrives, potentially involving pressure-responsive elements.

What are the optimal conditions for expressing recombinant P. profundum TrpD?

For expressing recombinant P. profundum TrpD, researchers should consider the following methodological approach:

• Expression System: E. coli BL21(DE3) or similar strains are commonly used for expressing recombinant proteins from bacterial sources. The use of plasmid-based systems, as demonstrated with the oriCII region of P. profundum expressed in E. coli , suggests a similar approach would be suitable for TrpD.

• Temperature: Since P. profundum is a psychrophilic to moderately piezophilic organism, expression at lower temperatures (15-20°C) may yield better results by allowing proper folding of the protein.

• Induction Conditions: Low IPTG concentrations (0.1-0.5 mM) and extended expression times (overnight) at reduced temperatures often improve the yield of soluble protein.

• Media Supplementation: Consider adding zinc (Zn²⁺) to the culture medium, as TrpD enzymes have been shown to utilize various metal ions including Zn²⁺ for enhanced activity .

These recommendations are based on extrapolation from successful expression strategies for other piezophilic proteins and related TrpD enzymes.

What purification strategies are most effective for recombinant P. profundum TrpD?

A multi-step purification strategy is recommended:

• Initial Capture: Immobilized metal affinity chromatography (IMAC) using a His-tag is effective for initial purification. Use a lysis buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 10 mM imidazole, with elution performed using an imidazole gradient (20-250 mM).

• Intermediate Purification: Ion exchange chromatography using either anion (Q-Sepharose) or cation (SP-Sepharose) exchangers, depending on the calculated pI of P. profundum TrpD.

• Polishing Step: Size exclusion chromatography using a Superdex 75 or 200 column to obtain highly pure protein and determine its oligomeric state.

• Buffer Considerations: Include Zn²⁺ (1-5 mM) in purification buffers if the enzyme shows metal dependency similar to other characterized TrpD enzymes .

• Storage Conditions: Store the purified enzyme in a buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM DTT, and 10% glycerol at -80°C to maintain stability.

This approach combines standard protein purification techniques with considerations specific to the properties of TrpD enzymes.

What are the key structural features of P. profundum TrpD?

Based on structural characteristics of characterized TrpD enzymes, P. profundum TrpD likely possesses:

• PRT Fold: The typical phosphoribosyltransferase fold with a small N-terminal α-helical domain and a larger C-terminal α/β domain .

• Conserved Motifs:

  • A KHGN motif (likely in positions similar to 101-104 in T. kodakaraensis TrpD) involved in anthranilate and PRPP binding

  • A conserved arginine residue (similar to Arg159 in T. kodakaraensis) essential for anthranilate binding and catalytic function

  • A highly conserved GTGGD motif, which is a signature sequence of the TrpD family involved in PRPP binding

• Metal Binding Sites: Potential zinc-binding motifs, possibly including a DE motif similar to the DE(217-218) identified in T. kodakaraensis TrpD .

• Oligomeric Structure: Likely forms a dimer in solution, with the dimerization potentially enhanced in the presence of metal ions like Zn²⁺ .

These structural features are critical for the catalytic function of the enzyme and may include special adaptations in P. profundum for high-pressure environments.

How might high-pressure adaptation affect the structure of P. profundum TrpD?

P. profundum as a piezophilic organism likely possesses TrpD with structural adaptations for high-pressure environments:

• Protein Packing: Potentially looser packing of the protein core compared to mesophilic homologs, allowing for greater compressibility under pressure.

• Surface Charge Distribution: Increased negative surface charge to maintain protein solubility under pressure.

• Flexibility in Active Site Region: Reduced rigidity in the active site to allow conformational changes necessary for catalysis under pressure.

• Metal Coordination: Possibly altered metal coordination geometry to maintain optimal catalytic activity under pressure. The zinc-binding properties observed in other TrpD enzymes might be modified to function optimally in high-pressure environments.

• Oligomeric Interface: Potentially strengthened subunit interactions to prevent pressure-induced dissociation of the likely dimeric structure.

These adaptations would represent evolutionary strategies to maintain enzyme function in the deep-sea environment where P. profundum naturally inhabits.

What are the kinetic parameters of P. profundum TrpD and how do they compare to other characterized TrpD enzymes?

While specific kinetic parameters for P. profundum TrpD are not provided in the search results, we can make educated predictions based on data from other TrpD enzymes:

P. profundum TrpD likely exhibits substrate inhibition by anthranilate at higher concentrations, similar to M. tuberculosis TrpD . The specific activity might be lower than that of hyperthermophilic enzymes due to the psychrophilic nature of P. profundum, but this would be compensated by higher catalytic efficiency at lower temperatures.

How does pressure affect the activity and stability of P. profundum TrpD?

As an enzyme from a piezophilic organism, P. profundum TrpD likely exhibits pressure-adapted functional properties:

• Pressure Optimum: The enzyme likely shows optimal activity at pressures corresponding to the deep-sea habitat of P. profundum (approximately 20-40 MPa), unlike enzymes from surface organisms that typically show decreased activity under pressure.

• Pressure Stability: Enhanced structural stability under high pressure compared to mesophilic homologs, potentially mediated by specific amino acid compositions and conformational flexibility.

• Pressure-Temperature Relationship: The enzyme might exhibit a coupled pressure-temperature relationship, with the temperature optimum shifting in response to pressure changes, a phenomenon observed in other proteins from piezophilic organisms.

• Catalytic Efficiency Under Pressure: Potentially maintains or even increases kcat/Km values under pressure, especially at lower temperatures characteristic of the deep sea.

• Ligand Binding Under Pressure: The binding affinity for substrates (anthranilate and PRPP) might be pressure-modulated, potentially with lower Km values under pressure.

These pressure-related properties would be critical for the function of P. profundum TrpD in its natural deep-sea environment and represent an important area for experimental investigation.

What metal ion dependencies does P. profundum TrpD exhibit?

Based on the metal ion dependencies observed in other TrpD enzymes, P. profundum TrpD likely shows the following characteristics:

• Primary Metal Dependency: Most TrpD enzymes utilize Mg²⁺ as the primary divalent cation for enzyme activity . P. profundum TrpD likely shares this requirement.

• Alternative Metal Utilization: Like TrpD from Salmonella typhimurium and Pectobacterium carotovorum, which can utilize Mn²⁺ and Co²⁺ in addition to Mg²⁺ , P. profundum TrpD might also show activity with alternative metal ions.

• Zinc Enhancement: Given the significant enhancement of activity observed with Zn²⁺ in T. kodakaraensis TrpD , P. profundum TrpD might similarly be activated by Zn²⁺, possibly through a conformational change mechanism or by facilitating dimerization.

• Metal Binding Sites: The enzyme likely contains specific metal binding motifs, potentially including a DE motif similar to DE(217-218) in T. kodakaraensis TrpD .

• Pressure-Metal Interaction: The metal preference might be pressure-dependent, with different metal ions providing optimal activity under different pressure conditions.

Experimental determination of these metal dependencies would provide valuable insights into the catalytic mechanism and evolutionary adaptations of P. profundum TrpD.

What assay methods are most suitable for measuring P. profundum TrpD activity?

Several complementary methods can be employed to assay P. profundum TrpD activity:

• Spectrophotometric Assay: Monitor the formation of PRA by measuring absorbance at 400 nm, where PRA exhibits a characteristic absorption peak. This can be performed in a reaction mixture containing:

  • 50 mM Tris-HCl, pH 7.5

  • 5 mM MgCl₂ (or alternative metal ions including ZnCl₂)

  • 0.5-4 μM anthranilate

  • 1 mM PRPP

  • Purified TrpD enzyme

• Fluorescence-Based Assay: Utilize the fluorescent properties of anthranilate (excitation 310 nm, emission 390 nm) and monitor the decrease in fluorescence as it is converted to PRA.

• High-Pressure Enzymatic Assay: To study the pressure dependence of activity, use specialized high-pressure equipment with optical windows that allow spectrophotometric measurements during pressurization.

• Coupled Enzyme Assay: Link the TrpD reaction to the next enzyme in the tryptophan biosynthesis pathway (PRA isomerase) and monitor the formation of downstream products.

• Radiolabeled Substrate Assay: For high sensitivity, use ¹⁴C-labeled anthranilate and measure the formation of labeled PRA by scintillation counting after separation.

These methods provide complementary approaches to characterize the enzymatic activity under various conditions, including different temperatures, pressures, pH values, and metal ion concentrations.

What techniques are available for studying the pressure adaptation mechanisms of P. profundum TrpD?

Several specialized techniques can be employed to investigate the pressure adaptation mechanisms:

• High-Pressure X-ray Crystallography: Determine the protein structure under various pressure conditions to identify pressure-induced conformational changes. This requires specialized high-pressure crystallography equipment.

• High-Pressure NMR Spectroscopy: Analyze the dynamic properties and structural changes of the enzyme under pressure using specialized high-pressure NMR cells.

• Pressure Perturbation Calorimetry: Measure the volumetric properties of the protein and its interactions with substrates under pressure to understand thermodynamic aspects of pressure adaptation.

• Site-Directed Mutagenesis: Create targeted mutations of residues predicted to be involved in pressure adaptation, followed by comparative activity assays under different pressure conditions.

• Molecular Dynamics Simulations: Perform in silico analysis of the enzyme's behavior under pressure, focusing on compressibility, water interaction networks, and conformational flexibility.

• Comparative Studies: Compare the properties of P. profundum TrpD with homologs from non-piezophilic organisms to identify specific adaptations. This could include chimeric protein construction and analysis.

These methodologies provide complementary approaches to unravel the molecular basis of pressure adaptation in P. profundum TrpD.

How does P. profundum TrpD contribute to the organism's piezophilic lifestyle?

The relationship between TrpD and piezophilic adaptation likely involves several interconnected mechanisms:

This multi-faceted relationship between TrpD function and piezophilic adaptation represents a rich area for further investigation.

How does the function of P. profundum TrpD relate to the organism's genomic structure, particularly its two-chromosome system?

The relationship between P. profundum TrpD function and the organism's genomic organization presents several intriguing research questions:

• Chromosomal Location: The trpD gene might be located on either chromosome I or II of P. profundum. If located on chromosome II, its replication would be regulated by the RctB protein, which has been shown to be essential for chromosome II replication and sensitive to high pressure .

• Coordinated Regulation: The expression of trpD might be coordinated with the replication cycle of its resident chromosome, potentially through shared regulatory elements.

• Horizontal Gene Transfer: The presence of trpD on chromosome II might reflect historical horizontal gene transfer events, potentially relating to adaptation to the deep-sea environment.

• Pressure-Responsive Expression: The chromosomal context of trpD might influence its expression under different pressure conditions, potentially through pressure-responsive regulatory elements or chromatin structure changes.

• Metabolic Integration: The distribution of tryptophan biosynthesis genes between the two chromosomes might reflect broader patterns of metabolic integration and regulation in P. profundum's adaptive strategy.

Investigating these relationships would provide insights into how genome architecture contributes to the piezophilic lifestyle of P. profundum.

What insights might comparative studies of P. profundum TrpD and TrpD from other Vibrionaceae provide about enzyme evolution under pressure?

Comparative studies could reveal evolutionary patterns in several key areas:

• Phylogenetic Analysis: Constructing a phylogenetic tree of TrpD sequences from Vibrionaceae inhabiting different depth niches could reveal pressure-related evolutionary trajectories and potential convergent adaptations.

• Signature Amino Acid Substitutions: Identification of amino acid substitutions unique to piezophilic species might reveal key residues involved in pressure adaptation, potentially clustering in functional domains or at protein interfaces.

• Selective Pressure Analysis: Calculating dN/dS ratios across the trpD gene in different Vibrionaceae lineages could identify regions under positive selection in piezophilic species.

• Structural Comparisons: Comparing protein structures (experimental or predicted) between piezophilic and non-piezophilic homologs might reveal changes in flexibility, compressibility, or active site architecture related to pressure adaptation.

• Functional Divergence: Characterizing the kinetic parameters, stability profiles, and metal dependencies of TrpD from Vibrionaceae spanning different depth ranges could establish correlations between these properties and habitat pressure.

These comparative approaches would provide insights not only into TrpD evolution but also into general principles of enzyme adaptation to high pressure.

What strategies can help overcome low expression yields of recombinant P. profundum TrpD?

When facing expression challenges, consider implementing these methodological solutions:

• Codon Optimization: P. profundum uses a different codon bias than common expression hosts like E. coli. Synthesizing a codon-optimized gene can significantly improve expression.

• Expression Host Selection: Beyond standard E. coli BL21(DE3), consider specialized strains like Rosetta (for rare codons), ArcticExpress (for cold-adapted expression), or C41/C43 (for potentially toxic proteins).

• Expression Tags and Fusion Partners: Test different affinity tags (His, GST, MBP) and their positions (N-terminal vs. C-terminal). MBP in particular can enhance solubility of challenging proteins.

• Induction Protocol Optimization:

  • Temperature: Try 12°C, 15°C, 20°C, and 25°C

  • IPTG concentration: Test a range from 0.1 mM to 1.0 mM

  • Induction time: Try 4h, 8h, overnight, and up to 48h at lower temperatures

  • Media: Compare rich (LB, TB) vs. minimal media with supplements

• Chaperone Co-expression: Co-express molecular chaperones (GroEL/ES, DnaK/J) to assist proper folding, particularly important for proteins from organisms with different thermal optima.

These approaches address different aspects of heterologous expression and can be systematically tested to identify optimal conditions.

How can researchers address potential metal ion contamination issues in P. profundum TrpD activity assays?

Metal ion contamination can significantly impact the interpretation of enzymatic assays, particularly for metalloenzymes like TrpD. Address this methodologically:

• Metal Chelation Treatment:

  • Treat buffers with Chelex-100 resin to remove metal contaminants

  • Include EDTA (1-5 mM) in initial purification steps, followed by extensive dialysis before activity assays

  • Use high-purity water (18.2 MΩ·cm) for all solutions

• Metal Analysis:

  • Perform ICP-MS analysis on purified enzyme to quantify bound metals

  • Use metal-specific fluorescent probes to detect trace metal contamination

• Controlled Metal Addition Experiments:

  • Test activity with systematically increasing concentrations of each relevant metal (Mg²⁺, Zn²⁺, Mn²⁺, Co²⁺)

  • Create a metal ion activity profile to identify true dependencies

• Buffer Component Considerations:

  • Avoid phosphate buffers when studying metal dependencies as they can chelate certain metal ions

  • Use HEPES or Tris buffers with caution, as they weakly bind some metal ions

• Control Experiments:

  • Include metal-free negative controls in all assays

  • Perform assays with known metal chelators (EDTA, EGTA, 1,10-phenanthroline) at various concentrations

These approaches will help establish genuine metal dependencies versus artifacts from contamination.

What approaches are effective when facing challenges in crystallizing P. profundum TrpD for structural studies?

When crystallization proves challenging, implement this systematic methodology:

• Protein Sample Preparation:

  • Ensure >95% purity by additional purification steps if needed

  • Check monodispersity by dynamic light scattering (DLS)

  • Try different buffer conditions (pH 5.5-8.5, various salts)

  • Remove flexible regions through limited proteolysis or construct design

  • Test different metal-bound states of the enzyme

• Crystallization Strategies:

  • Substrate/Product Complex: Co-crystallize with anthranilate, PRPP, or PRA to stabilize active conformation

  • Surface Engineering: Introduce surface mutations to reduce entropy (e.g., lysine methylation or Lys→Ala mutations)

  • Fusion Partners: Try crystallization chaperones like T4 lysozyme or MBP

  • Alternative Techniques: Consider LCP (lipidic cubic phase) or microseeding approaches

• Pressure Considerations:

  • Attempt crystallization under various pressures using specialized equipment

  • Include osmolytes known to mimic pressure effects (TMAO, glycine betaine)

• Alternative Structural Methods:

  • Cryo-EM for structure determination if crystallization remains problematic

  • SAXS to obtain low-resolution structural information

  • NMR for specific domain studies or dynamics investigations

• Orthologous Proteins:

  • Try crystallizing TrpD from closely related Photobacterium species

  • Create chimeric constructs with crystallizable TrpD proteins from other species

These methodical approaches address various aspects that might impede crystallization and provide alternatives when traditional crystallization fails.