Recombinant Photobacterium profundum Phosphopantetheine adenylyltransferase (coaD)

How does P. profundum PPAT structure differ from other bacterial homologs?

P. profundum PPAT shares the core structural features of the nucleotidyltransferase superfamily but exhibits adaptations associated with its deep-sea origin. Comparative analysis indicates that P. profundum PPAT contains:

• A dinucleotide-binding fold structurally similar to class I aminoacyl-tRNA synthetases

• Hexameric quaternary structure typical of bacterial PPATs

• Adaptations for functionality under high hydrostatic pressure

Although structural information specific to P. profundum PPAT is limited in the available literature, insights can be drawn from homologous bacterial PPATs, such as those from Mycobacterium and E. coli. For instance, the M. abscessus PPAT crystal structure reveals that substrate binding causes "a vice-like movement of active site residues lining the active site surface," and binding at one site influences the hexamer in a highly concerted manner .

Unlike eukaryotic systems where the final two steps of CoA biosynthesis are catalyzed by a single bifunctional enzyme (CoA synthase) containing a PPAT-like domain, bacterial PPATs function as standalone enzymes, making them attractive targets for antibiotic development .

What are the optimal conditions for heterologous expression of recombinant P. profundum PPAT?

For successful heterologous expression of recombinant P. profundum PPAT, researchers should consider the following optimized protocol based on similar deep-sea bacterial proteins:

Expression system construction:

• Amplify the coaD gene from P. profundum genomic DNA using specific primers targeting the complete open reading frame

• Clone the gene into an appropriate expression vector (e.g., pET28a SUMO vector with BamHI and HindIII restriction sites)

• Transform the construct into an E. coli expression strain capable of handling potentially toxic recombinant proteins

Optimal expression conditions:

• Host strains: E. coli BL21(DE3)Codonplus-RIL or Rosetta(DE3)pLysS

• Growth medium: LB medium in baffled flasks or P3 medium supplemented with 30 g/liter glucose in a fermentor

• Temperature: 15-30°C (lower temperatures may improve proper folding of psychrophilic proteins)

• Induction: 1 mM IPTG when A600 reaches 0.6 (flasks) or 2.5 (fermentor)

• Post-induction period: 3-16 hours

For P. profundum proteins, which naturally function under high pressure, expression at atmospheric pressure may affect protein folding and activity. Researchers should consider post-purification refolding or stabilization under conditions that mimic the native environment.

What purification strategies yield the highest purity and activity of recombinant P. profundum PPAT?

A multi-step purification strategy is recommended for obtaining high-purity, active recombinant P. profundum PPAT:

Initial extraction:

• Harvest cells by centrifugation (4,000 × g, 20 min, 4°C)

• Wash with 50 mM Tris-HCl buffer (pH 8.0)

• Resuspend cells in lysis buffer containing protease inhibitors

• Lyse cells using sonication or high-pressure homogenization

• Clarify lysate by centrifugation (20,000 × g, 30 min, 4°C)

Purification scheme:

• Metal affinity chromatography: For His-tagged constructs, use nickel or cobalt affinity columns with imidazole gradient elution

• Tag removal: If using a cleavable tag, digest with appropriate protease

• Size exclusion chromatography: For final polishing and buffer exchange

• Storage: Stabilize with 400 mM potassium glutamate and store at -80°C

The purified enzyme should be tested for activity using both forward and reverse reaction assays. For the forward reaction, incubate 1 mM 4'-phosphopantetheine with 0.1 mM ATP, 4 mM dithiothreitol, 2 mM MgCl₂, and purified enzyme in 50 mM Tris-HCl buffer (pH 8.0) containing 400 mM potassium glutamate. Monitor product formation by HPLC .

How do substrate specificity and kinetic parameters of P. profundum PPAT compare with other bacterial PPATs?

P. profundum PPAT exhibits substrate specificity and kinetic parameters that reflect its adaptation to the deep-sea environment. While specific kinetic data for P. profundum PPAT is limited in the literature, comparative analysis with other bacterial PPATs provides useful insights:

Substrate specificity:

• Primary substrates: 4'-phosphopantetheine and ATP

• The enzyme catalyzes the reversible reaction:
  4'-phosphopantetheine + ATP ⇌ dephospho-CoA + PPi

• The enzyme likely exhibits higher activity with 4'-phosphopantetheine than with structural analogs

Kinetic considerations:

• Temperature effects: As P. profundum is psychrophilic (cold-loving), its PPAT likely exhibits higher catalytic efficiency at lower temperatures (10-15°C) compared to mesophilic homologs

• Pressure effects: PPAT activity is likely optimized for high hydrostatic pressures (28 MPa), reflecting P. profundum's piezophilic nature

• Ion requirements: Requires Mg²⁺ as a cofactor, similar to other PPATs

Feedback inhibition:
Like other bacterial PPATs, P. profundum PPAT is likely subject to feedback inhibition by CoA, the end product of the pathway. This regulatory mechanism allows the cell to control CoA biosynthesis based on metabolic needs .

What are the catalytic mechanisms and transition states in the P. profundum PPAT reaction?

The catalytic mechanism of P. profundum PPAT follows the general reaction scheme observed in other bacterial PPATs, involving:

• Substrate binding: ATP and 4'-phosphopantetheine bind in the active site, with proper orientation facilitated by conserved residues

• Nucleophilic attack: The 4'-phosphate of phosphopantetheine performs a nucleophilic attack on the α-phosphate of ATP

• Transition state formation: Formation of a pentacovalent transition state at the α-phosphate

• Bond cleavage: The P-O bond between α and β phosphates of ATP is cleaved

• Product release: dephospho-CoA and pyrophosphate are released

Unlike many enzymes, PPAT does not involve direct covalent participation of active site residues. Instead, the enzyme functions by providing optimal positioning of substrates to reduce the activation energy of the transition state. This mechanism aligns with structural data showing:

• The nucleophilic 4'-phosphate of phosphopantetheine is positioned for in-line attack on the α-phosphate of ATP

• The enzyme orients the ATP β and γ-phosphates to stabilize the pentacovalent transition state

• Magnesium ions coordinate with the phosphate groups, facilitating the reaction

Ternary complex structures with bound substrates confirm this mechanism, as demonstrated in related PPATs where the 4'-phosphopantetheine substrate and the corresponding moiety in dephospho-CoA product overlap perfectly in the active site, with the α-phosphate positioned appropriately for nucleophilic attack .

What methodological approaches can be used to analyze P. profundum PPAT pressure adaptation?

To investigate P. profundum PPAT pressure adaptation, researchers can employ a multi-faceted approach:

Structural analysis under pressure:

• High-pressure X-ray crystallography: Determine structure under various pressures to observe conformational changes

• NMR spectroscopy: Analyze protein dynamics under pressure

• Small-angle X-ray scattering (SAXS): Monitor quaternary structure changes at different pressures

Functional characterization:

• High-pressure enzyme assays: Utilize pressure vessels to measure enzyme activity at different pressures

  • For example, prepare samples in thin-wall tubes and incubate in pressure vessels at various pressures (0.1-100 MPa)

  • Analyze reaction products by HPLC or coupled enzyme assays

• Thermal shift assays under pressure: Determine protein stability at different pressure-temperature combinations

• Isothermal titration calorimetry (ITC): Measure substrate binding parameters under various pressures

Comparative genomics and mutagenesis:

• Compare PPAT sequences from piezophilic (pressure-loving) and non-piezophilic bacteria to identify potential pressure-adaptive mutations

• Generate site-directed mutants targeting residues that differ between piezophilic and non-piezophilic PPATs

• Assess the effect of these mutations on pressure tolerance and enzymatic activity

These approaches would provide insights into the molecular basis of PPAT adaptation to high pressure in P. profundum and potentially reveal general principles of enzyme pressure adaptation.

How can P. profundum PPAT be utilized as a model for studying enzyme adaptation to extreme environments?

P. profundum PPAT represents an excellent model for studying enzyme adaptation to extreme environments, particularly to high hydrostatic pressure and low temperature. Research applications include:

Evolutionary adaptation studies:

• Comparative analysis of PPAT sequences from bacteria inhabiting different depths to identify adaptive mutations

• Reconstruction of ancestral PPAT sequences to trace the evolutionary path of pressure adaptation

• Correlation of structural features with habitat depth and pressure

Structure-function relationship under extreme conditions:

• Identification of structural elements that maintain function under high pressure

• Analysis of protein dynamics across a range of pressures

• Investigation of how pressure affects enzyme-substrate interactions and catalytic efficiency

Methodological framework:

• Express recombinant PPATs from organisms adapted to different depths

• Characterize their kinetic parameters under varying pressures and temperatures

• Perform structural analysis to correlate functional differences with structural features

• Use site-directed mutagenesis to introduce or remove adaptive features

• Test chimeric enzymes combining domains from piezophilic and non-piezophilic PPATs

This approach would provide insights not only into PPAT adaptation but also into general principles of protein adaptation to extreme environments, with potential applications in protein engineering and biotechnology.

What are the implications of P. profundum PPAT research for antibiotic development?

Research on P. profundum PPAT has significant implications for antibiotic development, particularly for deep-sea bacteria and related pathogens:

PPAT as an antibiotic target:

• PPAT catalyzes a rate-limiting step in the essential CoA biosynthetic pathway

• The coaD gene has been shown to be essential for growth in various bacteria, including mycobacteria

• Structural differences between bacterial and human PPAT-like domains facilitate target-specific antibiotic development without inducing mechanism-based toxicity

Drug discovery approaches:

• Structure-based drug design: Crystal structures of bacterial PPATs provide templates for rational design of inhibitors

• Fragment-based screening: Identify small-molecule fragments that bind to specific sites on PPAT

  • Thermal shift assays can be used to identify initial hits

  • X-ray crystallography and isothermal titration calorimetry (ITC) can validate and quantify binding

  • From such screening, compounds with binding affinities in the low micromolar range (Kd 3.2 ± 0.8 µM) have been identified for related PPATs

• Natural product screening: Test compounds from marine organisms for PPAT inhibition

Challenges and considerations:

• Penetration: Inhibitors must penetrate the bacterial cell envelope

• Resistance mechanisms: Bacteria possess intrinsic resistance mechanisms, including efflux pumps

• Selectivity: Inhibitors must selectively target bacterial PPAT over human PPAT-like domains

• Spectrum: Differences between PPATs from different bacterial species may affect inhibitor efficacy

The study of P. profundum PPAT provides insights into PPAT function under extreme conditions, potentially revealing unique inhibition strategies that could be applied to related pathogenic bacteria.

How can researchers overcome the challenges of expressing and characterizing enzymes from piezophilic organisms?

Expressing and characterizing enzymes from piezophilic organisms like P. profundum presents unique challenges. Here are methodological approaches to address these issues:

Expression challenges and solutions:

• Codon optimization: Adapt codon usage to the expression host (e.g., E. coli)

• Expression temperature: Lower expression temperature (15-20°C) to improve folding of psychrophilic proteins

• Specialized expression hosts: Use hosts adapted for cold-temperature expression or engineered for pressure tolerance

• Fusion tags: Employ solubility-enhancing tags such as SUMO, MBP, or thioredoxin

• Cell-free expression systems: Consider cell-free systems that can be operated under high pressure

Stabilization strategies:

• Buffer optimization: Include stabilizing agents such as potassium glutamate (400 mM) or glycerol

• Ligand stabilization: Co-purify with substrates or product analogs to enhance stability

• Pressure adaptation: Refolding or post-purification treatment under pressure to achieve native conformation

Characterization under native conditions:

• High-pressure equipment: Utilize pressure vessels adapted for spectroscopic or enzymatic assays

• In-situ monitoring: Develop methods for real-time activity monitoring under pressure

• Temperature control: Maintain appropriate low temperatures (10-15°C) during experiments

Case study on thermal stabilization:
Research on related proteins has shown that ligand binding can significantly enhance thermal stability. For instance, binding of dephospho-CoA to a PPAT from a thermophilic archaeon increased the apparent Tm from 76°C to 82°C as measured by differential scanning microcalorimetry . Similar approaches may be applicable to stabilize P. profundum PPAT during purification and characterization.

What are the current methodological limitations in studying P. profundum PPAT and how might they be addressed?

Current research on P. profundum PPAT faces several methodological limitations that need innovative solutions:

Limitation 1: High-pressure enzymatic assays

• Challenge: Standard enzymatic assays are difficult to perform under high pressure

• Solutions:

  • Develop miniaturized assays compatible with pressure vessels

  • Design coupled enzyme systems that function under pressure

  • Implement stopped-flow techniques with rapid decompression for sampling

  • Utilize spectroscopic methods that can penetrate pressure vessels

Limitation 2: Structural analysis under pressure

• Challenge: Obtaining structural information under native high-pressure conditions

• Solutions:

  • Adapt X-ray crystallography for high-pressure conditions

  • Employ computational modeling to predict pressure effects on structure

  • Use high-pressure NMR for solution-state structural analysis

  • Develop high-pressure cryo-EM techniques

Limitation 3: Heterologous expression

• Challenge: Achieving proper folding in non-piezophilic expression hosts

• Solutions:

  • Engineer expression hosts for pressure tolerance

  • Develop high-pressure fermentation systems

  • Optimize refolding protocols under pressure

  • Express in psychrophilic hosts that may better handle cold-adapted proteins

Limitation 4: Functional validation in native conditions

• Challenge: Confirming the physiological relevance of in vitro findings

• Solutions:

  • Develop genetic tools for P. profundum that function under pressure

  • Create reporter systems to monitor PPAT activity in vivo

  • Implement systems biology approaches to understand the role of PPAT in the context of pressure adaptation

  • Utilize comparative proteomics to identify pressure-responsive networks involving PPAT