Recombinant Photobacterium profundum Quinolinate synthase A (nadA)

What is Photobacterium profundum Quinolinate synthase A (nadA)?

Quinolinate synthase A (NadA) from Photobacterium profundum SS9 is a [4Fe-4S] cluster-containing enzyme that catalyzes a key step in the NAD de novo biosynthesis pathway I. This enzyme is responsible for the condensation of dihydroxyacetone phosphate (DHAP) and iminoaspartate to form quinolinic acid (QA), which serves as the universal precursor for NAD biosynthesis . P. profundum SS9 is a Gram-negative piezophilic bacterium originally isolated from the Sulu Sea, with its genome consisting of two chromosomes and an 80 kb plasmid .

What are the optimal growth conditions for Photobacterium profundum?

P. profundum SS9 exhibits adaptability to various pressure conditions but grows optimally at 28 MPa and 15°C. Importantly, its ability to grow at atmospheric pressure (0.1 MPa) makes it an ideal model organism for studying piezophily, as it allows for both easy genetic manipulation and culture . For laboratory culture, P. profundum SS9 is typically grown anaerobically at 17°C in marine broth supplemented with 20 mM glucose and 100 mM HEPES buffer (pH 7.5) .

How does the structure of P. profundum NadA compare to NadA from other organisms?

The structure of P. profundum NadA shares significant similarity with other prokaryotic NadA enzymes. Based on AlphaFold predictions, P. profundum NadA (UniProt: Q6LP50) shows a high confidence model (global pLDDT score of 93.85), suggesting a well-defined structure similar to characterized NadA proteins . Comparative analysis with NadA from Mycobacterium tuberculosis H37Rv (global pLDDT score: 93.97) indicates conservation of key structural elements across bacterial species . Crystal structures of related NadA proteins, such as that from Pyrococcus horikoshii, have revealed that the [4Fe-4S] cluster contains a unique non-cysteinyl-ligated iron ion (Fe a) that plays a crucial role in product binding .

What analytical methods can be used to assess the activity of recombinant P. profundum NadA?

Spectrophotometric Assay:
NadA activity can be measured using a coupled enzyme assay system monitoring NADH formation at 340 nm . The reaction mixture typically contains:

• 50 mM Tris-HCl buffer (pH 7.5)

• 1 mM iminoaspartate (or L-aspartate with L-aspartate oxidase)

• 1 mM DHAP

• 1 mM DTT

• 1-5 μg purified NadA enzyme

• Coupling enzymes for NADH detection

HPLC-Based Assay:
For direct quantification of quinolinate production:

• Prepare reaction mixture with 0.2 mM NMN and purified enzyme

• Incubate at 30°C for desired time points

• Terminate reaction by heat inactivation (95°C, 5 min)

• Analyze by HPLC using a C18 column with appropriate mobile phase

• Monitor product formation at 260-280 nm

Kinetic Parameter Determination:
For accurate kinetic characterization, vary substrate concentrations and determine initial reaction velocities. Calculate Km and kcat values using Michaelis-Menten equations .

How does pressure affect the expression and activity of NadA in P. profundum?

Proteomic analysis of P. profundum grown at different pressures reveals significant pressure-dependent regulation of metabolic pathways that likely impact NadA function and expression . When comparing atmospheric pressure (0.1 MPa) to high pressure (28 MPa) growth conditions:

Observed Pressure Effects:

• At high pressure (28 MPa), proteins involved in glycolysis/gluconeogenesis pathways are up-regulated, potentially increasing the availability of DHAP, a substrate for NadA .

• At atmospheric pressure (0.1 MPa), several proteins involved in oxidative phosphorylation are up-regulated, suggesting altered NAD/NADH utilization under different pressure conditions .

• Various transporters, including those involved in nutrient transport and assimilation, are differentially regulated by pressure, which may indirectly affect NadA function through metabolite availability .

Methodological Approach for Studying Pressure Effects:
To investigate pressure effects on recombinant NadA activity:

• Culture P. profundum SS9 anaerobically at 17°C in marine broth at different pressures (0.1 MPa and 28 MPa)

• Harvest cells in late exponential phase

• Extract proteins and analyze by shotgun proteomics or targeted Western blot

• Compare NadA expression levels between pressure conditions

• Perform in vitro enzymatic assays with purified NadA under various pressure conditions using specialized high-pressure equipment .

What molecular mechanisms underlie pressure adaptation in P. profundum NadA?

The molecular basis for pressure adaptation in P. profundum NadA likely involves structural modifications that maintain enzyme function under high hydrostatic pressure conditions:

Key Adaptation Mechanisms:

• Protein Flexibility Adjustments: Computational structure analysis suggests P. profundum NadA may contain regions with altered flexibility compared to non-piezophilic homologs, enabling maintenance of catalytic activity under pressure .

• Iron-Sulfur Cluster Stability: The [4Fe-4S] cluster in NadA, which is essential for catalysis, requires specific adaptations to remain stable under high pressure. These may include modified ligand interactions and protective structural elements .

• Active Site Architecture: The active site of NadA must accommodate substrate binding and product release under variable pressure conditions. The structure reveals that N1 and the C7 carboxylate group of quinolinic acid ligate to the unique iron ion (Fe a) in a bidentate fashion, which may be important for pressure adaptation .

• Conserved Residues: Three strictly conserved amino acids (equivalent to Glu198, Tyr109, and Tyr23 in related NadA structures) are in close proximity to the bound product. Substitution of these amino acids leads to complete loss of activity, indicating their essential role in catalysis regardless of pressure conditions .

Can P. profundum NadA be engineered for enhanced stability or activity?

Based on structural and functional data from related NadA enzymes, several approaches can be employed to engineer P. profundum NadA for enhanced properties:

Site-Directed Mutagenesis Strategies:

• Active Site Modifications: The A84L mutation in related quinolinate synthases has been studied for its effects on catalysis and could be explored in P. profundum NadA to alter substrate specificity or reaction rates .

• [4Fe-4S] Cluster Environment: Mutations of residues surrounding the iron-sulfur cluster could enhance stability under oxidative conditions, potentially increasing enzyme half-life.

• Domain Interface Engineering: Targeted modifications at domain interfaces might improve conformational stability while maintaining the essential dynamics required for catalysis.

Enzyme Complex Formation Approaches:
Creating fusion proteins or engineered enzyme complexes has shown promise for enhancing enzymatic pathways:

• Fuse NadA with its upstream enzyme L-aspartate oxidase (NadB) using optimized linkers such as (GS)6 or (G4S)2 to facilitate substrate channeling .

• Co-express NadA with chaperones like GroEL/GroES, which have been shown to be differentially regulated under pressure in P. profundum .

What experimental approaches can be used to study the reaction mechanism of NadA?

Understanding the catalytic mechanism of NadA requires sophisticated experimental techniques:

Spectroscopic Methods:

• EPR Spectroscopy: Characterize the [4Fe-4S] cluster and monitor changes during catalysis.

• Hyperfine Sublevel Correlation (HYSCORE) Spectroscopy: Confirm binding modes between substrates/products and the iron-sulfur cluster, as demonstrated with related NadA proteins .

• Mössbauer Spectroscopy: Analyze the electronic state of the iron atoms in the cluster.

Structural Approaches:

• X-ray Crystallography: Obtain high-resolution structures of P. profundum NadA in complex with substrates, intermediates, or inhibitors.

• Cryo-EM: Visualize conformational changes during the catalytic cycle.

Computational Methods:

• Molecular Dynamics Simulations: Investigate the effects of pressure on enzyme dynamics and substrate binding.

• QM/MM Calculations: Elucidate electronic aspects of the reaction mechanism involving the [4Fe-4S] cluster.

Kinetic and Mechanistic Studies:

• Stopped-flow Techniques: Monitor rapid reactions and identify transient intermediates.

• Isotope Labeling: Use deuterium or 13C-labeled substrates to track atom movements and determine rate-limiting steps.

• pH and Pressure Dependence: Characterize how pH and pressure affect reaction rates to identify key catalytic residues and conformational changes.

How does NadA interact with other enzymes in the NAD biosynthetic pathway?

NadA functions within the context of the NAD biosynthetic pathway, which requires coordinated activity of multiple enzymes:

Pathway Integration:

• NadA accepts iminoaspartate produced by L-aspartate oxidase (NadB) and DHAP from glycolysis.

• The reaction product, quinolinic acid, is subsequently utilized by quinolinate phosphoribosyltransferase (NadC).

Experimental Approaches to Study Pathway Interactions:

• Co-immunoprecipitation (Co-IP): Identify physical interactions between NadA and other pathway enzymes.

• Enzyme Complex Construction: Create artificial fusion proteins of NadA with its pathway partners:

  • The fusion of NadA and NadB with linkers like (GS)6 has been attempted, though with limited success in some systems .

  • Alternative fusion designs may yield more effective enzyme complexes.

• Metabolic Engineering Applications:

  • Engineering of E. coli for quinolinic acid production has demonstrated the importance of optimizing the entire pathway:

  Strain	Genetic Modifications	QA Production (mg/L)
  Wild-type + pTrc-nadA-nadB	Overexpression of nadA and nadB	667
  FZ734 + pTrc-aspC-nadB-nadA	Optimized gene order	404
  FZ763 + pFZGNB190	Multiple pathway optimizations	3,700

  These data highlight the importance of system-level optimization when working with NadA and suggest potential approaches for studying the enzyme in its native context .

What are common challenges when working with recombinant P. profundum NadA?

Expression and Solubility Issues:

• NadA easily forms inclusion bodies when overexpressed, as observed in related systems .

• The [4Fe-4S] cluster is oxygen-sensitive and can degrade during purification.

Recommended Solutions:

• Lower induction temperature (16-18°C)

• Reduce IPTG concentration (0.1-0.2 mM)

• Co-express with iron-sulfur cluster assembly proteins

• Perform all steps under anaerobic conditions

• Include iron and sulfur sources in the growth medium

Activity Measurement Challenges:

• The substrate iminoaspartate is unstable and must be generated in situ .

• Direct detection of quinolinate can be difficult without specialized equipment.

Recommended Solutions:

• Use coupled enzyme assays for real-time monitoring

• Develop HPLC or LC-MS methods for product quantification

• Consider using stable substrate analogs for binding studies

How can researchers distinguish between the effects of pressure, temperature, and other environmental factors on NadA function?

Experimental Design Strategies:

• Factorial Experimental Design: Systematically vary pressure, temperature, pH, and ionic strength to identify individual and interactive effects on NadA activity.

• In vitro vs. In vivo Studies:

  • In vitro: Purify NadA and test under controlled conditions in pressure chambers

  • In vivo: Compare gene expression and metabolite profiles under different conditions

• Control Experiments:

  • Use pressure-insensitive enzymes as controls

  • Compare P. profundum NadA with homologs from non-piezophilic organisms

Data Analysis Approaches:

• Apply multivariate statistical methods to distinguish between environmental effects

• Develop mathematical models that account for multiple variables

• Use principal component analysis to identify the most significant factors affecting NadA function