Recombinant Photobacterium profundum NH (3)-dependent NAD (+) synthetase (nadE)

What is Photobacterium profundum nadE and what role does it play in bacterial metabolism?

Photobacterium profundum nadE encodes an NH(3)-dependent NAD(+) synthetase that catalyzes the final step in NAD biosynthesis, converting nicotinic acid adenine dinucleotide (NaAD) to nicotinamide adenine dinucleotide (NAD+) through an amidation reaction. This enzyme is essential for P. profundum, a deep-sea bacterium isolated from the Sulu Sea at a depth of 2.5 km . As a piezophile, P. profundum SS9 grows optimally at 28 MPa and 15°C .

NAD+ is a critical cofactor for numerous cellular redox reactions and signaling pathways. The nadE-catalyzed reaction requires ATP and ammonia as substrates, converting 1 molecule of ATP to AMP and PPi per cycle of amidation . This reaction represents the convergence point for both de novo and salvage pathways of NAD biosynthesis, explaining why nadE is highly conserved and essential across bacterial species .

How does P. profundum nadE differ structurally from other bacterial NAD synthetases?

P. profundum nadE belongs to the NH(3)-dependent class of NAD synthetases, which differs from glutamine-dependent NAD synthetases (NadE2Gln) that contain an additional glutamine amidotransferase (GAT) domain. The NH(3)-dependent enzymes are typically smaller as they lack this domain .

The active site of NAD synthetases contains two primary binding regions:

• A nicotinosyl binding site that accommodates the nicotinic acid moiety

• An adenosyl binding site that interacts with the adenine portion

Structural differences between various bacterial NAD synthetases determine their substrate preferences. For example, in Francisella tularensis, a divergent member of the NadE family shows a strong preference for NaMN over NaAD as substrate due to specific amino acid substitutions in the binding pocket . These structural differences include:

• Altered residues in the nicotinosyl binding site (replacement of glycine and arginine residues with glutamine and tryptophan)

• Significant movements in the α9 helix (≈3.5 Å) and the α5 helix (≈1.0 Å)

• Replacement of valine by arginine to provide additional interactions with a free phosphoryl group

What are the most effective methods for expressing and purifying recombinant P. profundum nadE?

The expression and purification of recombinant P. profundum nadE can be achieved through the following protocol:

• Cloning: The nadE gene should be PCR-amplified from P. profundum SS9 genomic DNA and cloned into an expression vector (commonly pET series) with an N-terminal 6xHis tag to facilitate purification .

• Expression conditions: Transform the construct into E. coli BL21(DE3) or a similar expression strain. Culture cells at a lower temperature (15-18°C) after induction to enhance proper folding of the psychrophilic enzyme .

• Purification steps:

  • Harvest cells and lyse using sonication in a buffer containing 50 mM HEPES (pH 7.5), 300 mM NaCl, 10% glycerol, and 10 mM imidazole

  • Purify using Ni-affinity chromatography with a gradient elution (10-250 mM imidazole)

  • Further purify by gel filtration chromatography using a Superdex 200 column

• Quality control: Assess purity by SDS-PAGE and confirm identity by mass spectrometry or Western blotting .

For optimal activity, the purified enzyme should be stored in a buffer containing reducing agents such as DTT (1-5 mM), as NAD synthetases often show enhanced activity in reducing environments .

What assays are available for measuring P. profundum nadE activity, and what are their relative advantages?

Several assays are available for measuring P. profundum nadE activity:

• Spectrophotometric coupled assay:

  • Principle: The NAD+ produced is reduced to NADH by alcohol dehydrogenase, which can be monitored at 340 nm

  • Components: NaAD substrate, ATP, NH4Cl (or glutamine), MgCl2, alcohol dehydrogenase, and ethanol

  • Advantages: Real-time monitoring, suitable for kinetic measurements

  • Limitations: Indirect measurement, potential interference from other enzymes

• HPLC-based assay:

  • Principle: Direct separation and quantification of substrate (NaAD) and product (NAD+)

  • Advantages: Direct measurement, higher specificity, can detect intermediates

  • Limitations: Lower throughput, requires specialized equipment

• Colorimetric coupled assay using glutamate dehydrogenase:

  • Principle: The NH3 released during the glutamine-dependent reaction is coupled to the reductive amination of α-ketoglutarate by glutamate dehydrogenase, consuming NADPH

  • Advantages: Can be performed in microtiter plates (200 μL scale), cost-effective

  • Limitations: Only applicable for glutamine-dependent enzymes

• Mass spectrometry:

  • Principle: Direct detection of substrates, products, and intermediates

  • Advantages: Highest specificity, can detect multiple analytes simultaneously

  • Limitations: Requires specialized equipment, typically lower throughput

Standard reaction conditions typically include:

• 50 mM HEPES buffer (pH 7.5)

• 2 mM ATP

• 1 mM NaAD

• 5 mM NH4Cl (or glutamine)

• 10 mM MgCl2

• 37°C incubation (or lower temperature for P. profundum enzyme)

What methodologies can be used to study nadE function under different pressure conditions?

Studying nadE function under different pressure conditions requires specialized equipment and approaches:

• High-pressure bioreactors:

  • Stainless steel pressure vessels with temperature control (typically water-cooled)

  • Cultures grown in sealed, flexible containers (like Pasteur pipettes) to ensure even pressure distribution

  • Operating range of 0.1-90 MPa to cover the P. profundum growth range

• Real-time growth monitoring:

  • Modified microplate readers equipped with pressure chambers

  • Monitoring absorbance at 600 nm to track bacterial growth under pressure

  • Care must be taken to ensure anaerobic conditions during pressure cultivation

• Activity assays under pressure:

  • Specialized high-pressure stopped-flow devices for enzyme kinetics

  • Fluorescence-based assays that can be measured through sapphire windows in pressure chambers

  • Pressure may affect assay parameters, requiring careful baseline controls

• Structural analysis under pressure:

  • High-pressure nuclear magnetic resonance (NMR)

  • High-pressure X-ray crystallography

  • Molecular dynamics simulations incorporating pressure effects

• Comparative approaches:

  • Parallel analysis of nadE from pressure-sensitive and pressure-adapted organisms

  • Site-directed mutagenesis to identify pressure-sensing residues

  • Construction of chimeric enzymes to determine pressure-adaptation domains

For accurate results, all reagents should be pre-equilibrated to the test pressure before initiating reactions, and multiple biological and technical replicates should be performed to account for pressure-related variability .

What are the kinetic parameters of P. profundum nadE and how do they compare to other bacterial NAD synthetases?

The kinetic parameters of P. profundum nadE reflect its adaptation to deep-sea conditions. Although specific kinetic data for P. profundum nadE is limited in the literature, comparable NAD synthetases provide insight into expected parameters:

NadE is a divergent member of the NadE family with NMN synthetase activity

Key observations about NAD synthetases:

• Substrate specificity:

  • Canonical NAD synthetases (like B. anthracis NadE) show strong preference for NaAD over NaMN (>500-fold higher catalytic efficiency)

  • Divergent NadE enzymes (like F. tularensis NadE*) show preference for NaMN over NaAD (~60-fold higher catalytic efficiency)

• Cofactor requirements:

  • All NAD synthetases require ATP as a cofactor

  • Mg²⁺ is essential for activity, forming coordination complexes at the ATP-binding site

  • A second Mg²⁺ position closer to the NaAD-binding site has been observed at pH 7.5, which may be catalytically active

• Environmental adaptations:

  • Cold-adapted enzymes like those from P. profundum typically show higher kcat and higher Km values at lower temperatures compared to mesophilic counterparts

  • Pressure affects enzyme kinetics by influencing protein conformational dynamics

P. profundum nadE likely shows optimal activity at lower temperatures (10-15°C) and higher pressures (28 MPa) consistent with its native deep-sea environment .

How is P. profundum nadE activity regulated, and what factors affect its catalytic function?

P. profundum nadE activity is regulated through multiple mechanisms:

• Feedback inhibition:

  • NAD+ acts as a feedback inhibitor of NAD synthetase

  • Studies with other bacterial NAD synthetases show that NAD+ inhibition is non-competitive with respect to both NaAD and glutamine substrates

  • This inhibition changes Vmax but not Km, indicating allosteric regulation

• Redox state:

  • NAD synthetases, particularly glutamine-dependent forms, show enhanced activity in reducing environments

  • DTT increases activity when glutamine is the nitrogen donor, suggesting the importance of reduced cysteine residues in the catalytic mechanism

  • This effect is not observed when ammonia is used directly as the nitrogen donor

• Protein-protein interactions:

  • In some bacteria, regulatory proteins can modulate NAD synthetase activity

  • For example, in Azospirillum brasilense, the PII protein GlnZ can relieve NAD+ inhibition of NadE2

  • These interactions are often regulated by effector molecules like ATP, ADP, and 2-oxoglutarate

• Conformational changes:

  • Binding of substrates induces significant conformational changes in NAD synthetases

  • Two flexible loops (82-87 and 204-225) are stabilized by ATP binding

  • The major loop (204-225) has roles in substrate recognition, stabilization, and protection of reaction intermediates

• Post-translational modifications:

  • Phosphorylation sites have been identified in human NMNAT, suggesting similar regulation might occur in bacterial systems

  • These modifications may modulate activity or interactions with other proteins

Under high pressure conditions, P. profundum likely has additional regulatory mechanisms that maintain enzyme activity and stability, possibly through pressure-sensitive residues or domains that undergo conformational changes with pressure .

What genomic context surrounds the nadE gene in P. profundum, and how does this inform our understanding of NAD metabolism?

The genomic context of nadE in P. profundum provides important insights into NAD metabolism and evolutionary adaptations:

• Genomic organization:

  • P. profundum has two circular chromosomes and an 80 kb plasmid

  • The nadE gene is likely located on chromosome 1, as part of the core genome conserved among Vibrionaceae

  • Surrounding genes often include:

    • Genes involved in NAD biosynthesis (nadD, pncA, pncB)

    • Genes related to DNA repair and recombination (recD, recB)

    • General stress response genes

• NAD biosynthetic pathways in P. profundum:

  • Contains genes for de novo synthesis from aspartate (nadA, nadB, nadC)

  • Has salvage/recycling pathways via nicotinamide (pncA, pncB)

  • The nadD and nadE genes form the universal downstream pathway converting NaMN to NAD+

  • May lack the amidated salvage pathways seen in some bacteria (nadV, nadM)

• Comparative genomics:

  • Unlike some bacteria that have NadR homologs enabling alternate NAD synthesis routes, Photobacterium species rely on the classic NadD/NadE pathway

  • The genome contains multiple rRNA operons with high levels of variation, believed to help P. profundum rapidly respond to changes in pressure

• Transcriptional regulation:

  • RNA-seq analysis of P. profundum reveals complex transcriptional responses to pressure

  • NAD metabolism genes may be co-regulated with stress response genes under pressure changes

  • The ToxR transcriptional regulator, important in Vibrionaceae, may influence expression of NAD metabolism genes

This genomic context places P. profundum nadE within a pressure-responsive metabolic network, highlighting its importance in the adaptation of this bacterium to its deep-sea environment .

What structural features of P. profundum nadE contribute to its function under high-pressure conditions?

While specific structural data for P. profundum nadE is not fully characterized, several features likely contribute to its function under high-pressure conditions:

• Amino acid composition adaptations:

  • Increased proportion of charged residues on protein surfaces to maintain hydration under pressure

  • Decreased hydrophobic core volume to minimize pressure-induced destabilization

  • Reduction in the number of cavities within the protein structure

• Loop regions and flexibility:

  • Modified loop regions that maintain necessary flexibility under pressure

  • Potentially altered dynamics of the catalytic loops (similar to loops 82-87 and 204-225 identified in other NAD synthetases)

  • Strategic placement of glycine residues to allow conformational changes under pressure

• Active site architecture:

  • Coordination of Mg²⁺ ions that stabilize the ATP binding site

  • The position of a second catalytic Mg²⁺ ion may be optimized for activity under pressure

  • Modified substrate binding pockets that accommodate pressure-induced changes in substrate conformation

• Oligomeric structure:

  • NAD synthetases typically form dimers, and the dimer interface may contain adaptations to maintain stability under pressure

  • Pressure affects protein-protein interactions, so interface residues likely show pressure-specific adaptations

• Co-factor binding sites:

  • ATP binding induces conformational changes in loop regions

  • These regions may have evolved to maintain optimal conformation under high pressure

  • The adenine base of ATP forms hydrogen bonds with the minor loop (82-87), which could be modified in P. profundum to function optimally under pressure

These adaptations would allow P. profundum nadE to maintain catalytic activity at the high pressures found in its deep-sea habitat while preserving sufficient stability and flexibility for function .

How does the substrate specificity of P. profundum nadE compare with divergent NadE family members like F. tularensis NMN synthetase?

The substrate specificity of NAD synthetase family members reveals important evolutionary adaptations:

• Canonical NAD synthetases vs. divergent members:

  • Canonical NAD synthetases (like those in B. anthracis) strongly prefer NaAD over NaMN (>500-fold preference)

  • Divergent members (like F. tularensis NadE*) show preference for NaMN over NaAD (~60-fold preference)

  • P. profundum nadE likely follows the canonical pattern, preferring NaAD as substrate

• Structural basis for substrate specificity:

  • Comparing F. tularensis NadE* with B. anthracis NadE reveals key structural differences:

    • Replacement of glycine and arginine by glutamine and tryptophan in the nicotinosyl binding site

    • Movement of α9 helix (~3.5 Å) and α5 helix (~1.0 Å) creating a tighter binding pocket

    • Replacement of valine by arginine providing additional interactions with the phosphoryl group of NaMN

• Evolutionary implications:

  • Divergent substrate preferences reflect different NAD biosynthetic pathways:

    • Classic Route I: NaMN → NaAD → NAD+ (requires NadD and canonical NadE)

    • Alternative Route II: NaMN → NMN → NAD+ (requires NMN synthetase and NMN adenylyltransferase)

  • These alternative routes may provide metabolic flexibility or evolutionary advantages in specific environments

• Environmental adaptations:

  • High pressure may influence substrate binding and specificity

  • P. profundum nadE might show pressure-dependent changes in substrate preference

  • Deep-sea adaptations could include modified substrate binding pockets that maintain optimal configuration under pressure

The comparison between P. profundum nadE and divergent family members provides insights into the evolution of NAD biosynthesis pathways and adaptation to extreme environments .

What role might P. profundum nadE play in the bacterium's response to combined pressure and temperature stress?

P. profundum nadE likely plays a multifaceted role in the bacterium's response to combined pressure and temperature stress:

• Metabolic regulation:

  • NAD+/NADH balance is critical for redox homeostasis under stress conditions

  • P. profundum differentially regulates metabolic pathways in response to pressure:

    • Glycolysis/gluconeogenesis proteins are up-regulated at high pressure

    • Oxidative phosphorylation proteins are up-regulated at atmospheric pressure

  • NadE activity may be coordinated with these metabolic shifts to maintain appropriate NAD+ levels

• Integration with stress response systems:

  • P. profundum up-regulates stress response genes (htpG, dnaK, dnaJ, groEL) at atmospheric pressure

  • Interestingly, proteomic studies show that GroEL and DnaK are up-regulated at high pressure, while DnaJ is down-regulated

  • NAD+ metabolism may be coordinated with these stress responses through regulatory networks

• Membrane composition adaptation:

  • P. profundum alters membrane fatty acid composition in response to pressure and temperature

  • Increased proportions of monounsaturated (MUFAs) and polyunsaturated fatty acids (PUFAs) are observed at decreased temperature or elevated pressure

  • NAD+ metabolism supports fatty acid biosynthesis and may be coordinated with these changes

• Transcriptional regulation:

  • RNA-seq analysis reveals complex transcriptional responses to pressure

  • NAD+ levels can influence the activity of sirtuins and other NAD+-dependent regulators

  • The nadE gene may be part of a pressure-responsive regulon

• Protein-protein interactions:

  • Under stress conditions, NAD synthetase may interact with stress response proteins

  • These interactions could be modulated by post-translational modifications

  • High pressure and low temperature may alter these interaction networks

The integrated response to combined pressure and temperature stress likely involves coordinated regulation of nadE with other metabolic and stress response systems to maintain cellular homeostasis in the deep-sea environment .

How can recombinant P. profundum nadE be utilized as a model for understanding deep-sea enzyme adaptations?

Recombinant P. profundum nadE serves as an excellent model system for understanding deep-sea enzyme adaptations:

• Comparative structural biology:

  • Comparison with mesophilic and thermophilic homologs can reveal specific adaptations

  • Crystallographic studies at different pressures can identify pressure-sensitive regions

  • Molecular dynamics simulations can predict conformational changes under pressure

• Directed evolution studies:

  • Starting with P. profundum nadE, directed evolution can be used to enhance or modify pressure adaptation

  • Selection under different pressure regimes can reveal alternative adaptive solutions

  • The identified mutations can provide insights into critical residues for pressure adaptation

• Chimeric enzyme construction:

  • Domains from P. profundum nadE can be swapped with those from pressure-sensitive homologs

  • Testing these chimeras under pressure can identify specific regions responsible for pressure adaptation

  • This approach can map pressure-sensing domains within the enzyme

• Structure-function relationship studies:

  • Site-directed mutagenesis targeting conserved residues can reveal their role in pressure adaptation

  • The effects of mutations on kinetic parameters under different pressures can be measured

  • These studies can distinguish between stabilizing vs. catalytic adaptations

• Pressure-adapted biotechnology applications:

  • Understanding pressure adaptations in P. profundum nadE could inform the design of pressure-resistant enzymes for industrial applications

  • Deep-sea adapted enzymes may have unique properties useful in high-pressure bioprocessing

  • The principles learned could be applied to engineer other enzymes for extreme environments

By using P. profundum nadE as a model, researchers can gain insights into the molecular basis of pressure adaptation, with implications for understanding deep-sea life and developing biotechnological applications .

What insights does P. profundum nadE provide for the development of antimicrobial strategies targeting NAD biosynthesis?

P. profundum nadE offers valuable insights for antimicrobial development:

• Target validation:

  • NAD biosynthesis enzymes NadD and NadE are validated as essential targets across bacteria

  • Depleting these enzymes has strong bactericidal effects in model organisms

  • The near-universal conservation of these enzymes makes them attractive targets

• Structural insights for inhibitor design:

  • Deep-sea adaptations in P. profundum nadE may reveal unique structural features

  • Comparing pressure-adapted and mesophilic NAD synthetases can identify:

    • Conserved catalytic residues as targets for broad-spectrum inhibitors

    • Variable regions that could be targeted for selective inhibition

• Metabolic consequences of inhibition:

  • Inhibiting NadE causes:

    • Complete depletion of NAD+/NADH pools

    • Accumulation of biosynthetic precursors (NaMN, NaAD)

    • Global changes in central carbon metabolism

    • These effects are bactericidal rather than bacteriostatic

• Resistance considerations:

  • Some bacteria possess alternative NAD biosynthetic routes

  • P. profundum likely relies solely on the canonical NadD/NadE pathway

  • Understanding route diversity helps predict potential resistance mechanisms

• Novel inhibition strategies:

  • Targeting allosteric sites identified in P. profundum nadE

  • Disrupting protein-protein interactions that regulate NAD synthetase activity

  • Developing combination strategies targeting multiple steps in NAD biosynthesis

• Selective toxicity:

  • Structural differences between bacterial and human NAD biosynthetic enzymes

  • Human NMNAT exhibits substantial structural differences from bacterial enzymes

  • These differences can be exploited to develop selective inhibitors

The study of P. profundum nadE contributes to understanding structural and functional diversity within NAD synthetases, informing strategies for developing novel antibiotics targeting this essential enzyme family .

What unexplored aspects of P. profundum nadE warrant further investigation?

Several important aspects of P. profundum nadE remain unexplored and warrant further investigation:

• Pressure-dependent conformational dynamics:

  • How does pressure affect the conformational states of P. profundum nadE?

  • Which regions of the enzyme are most sensitive to pressure changes?

  • How do these conformational changes affect catalytic efficiency?

• Regulatory networks:

  • Does P. profundum have pressure-sensing regulatory systems that modulate nadE expression?

  • Are there protein-protein interactions specific to P. profundum that regulate nadE activity?

  • How is nadE expression coordinated with other pressure-responsive genes?

• Post-translational modifications:

  • Are there pressure-dependent post-translational modifications of P. profundum nadE?

  • Do these modifications affect enzyme activity, stability, or interactions?

  • What kinases or other modifying enzymes regulate nadE function?

• Alternative substrates and reaction pathways:

  • Does P. profundum nadE show activity with alternative substrates under high pressure?

  • Are there pressure-dependent changes in substrate specificity?

  • Could P. profundum utilize alternative NAD biosynthetic routes under specific conditions?

• Evolutionary origins of pressure adaptation:

  • What evolutionary path led to pressure adaptation in P. profundum nadE?

  • Are the pressure adaptations in nadE similar to those in other P. profundum enzymes?

  • How do the adaptations in P. profundum compare to those in other piezophiles?

• Role in bacterial survival under extreme conditions:

  • How does nadE function contribute to P. profundum survival under combined stresses?

  • Is nadE activity limiting for growth under certain pressure/temperature combinations?

  • How does NAD+ availability influence global gene expression under pressure?

Addressing these questions will require interdisciplinary approaches combining structural biology, biochemistry, genetics, and computational methods adapted for high-pressure conditions .

What technological advances would facilitate deeper understanding of P. profundum nadE function under native deep-sea conditions?

Advancing our understanding of P. profundum nadE under native deep-sea conditions requires innovative technological approaches:

• High-pressure structural biology techniques:

  • Development of high-pressure X-ray crystallography systems capable of operating at 28 MPa

  • Cryo-electron microscopy methods adapted for visualizing proteins under pressure

  • High-pressure NMR for studying protein dynamics in solution under deep-sea conditions

• In situ deep-sea sampling and analysis:

  • Autonomous underwater vehicles equipped with molecular biology capabilities

  • Pressure-retaining sampling devices that preserve native states of enzymes

  • In situ gene expression analysis tools to capture natural regulation patterns

• Advanced high-pressure bioreactors:

  • Continuous culture systems operating at high pressure with real-time monitoring

  • Microfluidic devices for single-cell analysis under pressure

  • Systems allowing rapid pressure transitions to study dynamic responses

• Computational approaches:

  • Enhanced molecular dynamics simulations incorporating accurate pressure effects

  • Machine learning algorithms to predict pressure-adaptive mutations

  • Systems biology models integrating metabolic, transcriptomic, and proteomic data under pressure

• High-throughput screening under pressure:

  • Miniaturized high-pressure systems for parallel enzyme activity assays

  • Fluorescence-based activity probes compatible with high-pressure vessels

  • Automated systems for screening enzyme variants under pressure

• Genetic manipulation tools optimized for piezophiles:

  • Pressure-resistant transformation systems

  • CRISPR-Cas9 systems optimized for function in P. profundum

  • Inducible gene expression systems functional across pressure ranges

• Metabolic labeling techniques:

  • Stable isotope probing compatible with high-pressure cultivation

  • Methods to track NAD+ turnover and utilization under pressure

  • Techniques to measure metabolic fluxes at the single-cell level under pressure