Recombinant Idiomarina loihiensis Probable inorganic polyphosphate/ATP-NAD kinase (ppnK)

What is Idiomarina loihiensis and why is it significant for enzyme research?

Idiomarina loihiensis is a γ-proteobacterium isolated from hydrothermal vents at a depth of 1,300 meters on the Lōihi submarine volcano near Hawaii. Unlike obligate anaerobic vent hyperthermophiles, I. loihiensis inhabits partially oxygenated cold waters at the periphery of hydrothermal vents and can survive in a wide range of temperatures (4°C to 46°C) and salinities (0.5% to 20% NaCl) . This remarkable environmental adaptability makes it an excellent source for studying enzymes that function under diverse conditions. The organism's genome consists of a single circular chromosome of 2,839,318 bp with an average G+C content of 47%, encoding 2,640 proteins . I. loihiensis represents a distinct lineage among γ-Proteobacteria, having branched from the main trunk of the γ-proteobacterial tree after the Pseudomonas lineage but before the Vibrio cluster .

What is the function of inorganic polyphosphate/ATP-NAD kinase (ppnK)?

NAD kinase (NADK) is a crucial enzyme that catalyzes the phosphorylation of NAD+ to produce NADP+, which serves as an essential cofactor in numerous metabolic reactions, particularly those involved in biosynthetic pathways and antioxidant defense systems . The enzyme can utilize either ATP or inorganic polyphosphate [poly(P)] as a phosphoryl donor, depending on its specificity . In γ-proteobacteria like Idiomarina loihiensis, NADKs are typically ATP-specific, meaning they preferentially use ATP rather than poly(P) as a phosphoryl donor . This specificity has important implications for the organism's metabolism and energy utilization strategies, particularly in the nutrient-limited deep-sea environment where I. loihiensis is found .

What is the difference between ATP-specific NADKs and poly(P)/ATP-NADKs?

The key difference lies in their phosphoryl donor specificity:

ATP-specific NADKs, like those naturally found in Idiomarina loihiensis, strongly prefer ATP as a phosphoryl donor and have limited ability to utilize poly(P) . In contrast, poly(P)/ATP-NADKs can efficiently use both ATP and inorganic polyphosphate. This difference is significant from an evolutionary perspective and has practical implications for biotechnological applications, particularly in the industrial production of NADP+ .

How is recombinant Idiomarina loihiensis ppnK typically expressed and purified?

Based on standard protocols for recombinant proteins from I. loihiensis, the expression and purification typically follow these steps:

• Cloning: The ppnK gene is amplified from I. loihiensis genomic DNA using PCR with specific primers containing appropriate restriction sites.

• Vector construction: The amplified gene is cloned into an expression vector (typically containing a His-tag or other affinity tag for purification).

• Transformation: The recombinant vector is transformed into a suitable E. coli expression strain.

• Protein expression: Cultures are grown to mid-log phase (OD600 ≈ 0.6) and then induced with IPTG (typically 0.1-1.0 mM) for 4-16 hours at temperatures between 16-37°C, depending on protein solubility.

• Cell lysis: Bacterial cells are harvested by centrifugation and disrupted using sonication or mechanical lysis in an appropriate buffer system.

• Purification: The recombinant protein is purified using affinity chromatography (such as Ni-NTA for His-tagged proteins), followed by additional purification steps like ion exchange or size exclusion chromatography if needed .

• Verification: Purity is typically assessed by SDS-PAGE (>85% purity is commonly achieved) and activity assays specific to NAD kinase .

How can the poly(P)-utilizing ability be conferred to the ATP-specific NADK from Idiomarina loihiensis?

Research has demonstrated that the ability to utilize poly(P) can be conferred to ATP-specific NADKs from γ-proteobacteria through a single amino acid substitution . This transformation involves identifying and modifying a key residue that determines phosphoryl-donor specificity.

The methodology involves:

• Sequence alignment: Align the amino acid sequences of ATP-specific NADKs with poly(P)/ATP-NADKs to identify conserved and variable regions.

• Structural analysis: Examine crystal structures (when available) to identify residues involved in phosphoryl donor binding.

• Site-directed mutagenesis: Create point mutations at candidate residues that might alter phosphoryl donor specificity.

• Activity assays: Test the mutant enzymes for their ability to utilize both ATP and poly(P) as phosphoryl donors.

Research has shown that this single amino acid substitution is sufficient to create a functional poly(P)/ATP-NADK from an ATP-specific enzyme . The specificity determinant residue appears to be highly conserved among γ-proteobacterial NADKs, suggesting an evolutionary significance to this feature .

What are the kinetic parameters of wild-type and engineered variants of Idiomarina loihiensis ppnK?

While specific kinetic parameters for I. loihiensis ppnK are not directly provided in the search results, comparable data from related enzymes can provide insight into expected values. For NADKs, typical kinetic parameters include:

To determine these parameters experimentally, researchers would:

• Measure initial velocities at varying substrate concentrations

• Plot the data using Lineweaver-Burk, Eadie-Hofstee, or non-linear regression methods

• Calculate Km, Vmax, and kcat values for both ATP and poly(P) as phosphoryl donors

• Compare the catalytic efficiency (kcat/Km) for both substrates

These analyses would reveal the degree to which the engineered variant has acquired poly(P) utilization capability while maintaining its original ATP-dependent activity .

How does the crystal structure of Idiomarina loihiensis ppnK compare to other NADKs?

While the specific crystal structure of I. loihiensis ppnK is not available in the provided search results, a comparative analysis can be inferred from related structures like the Inorganic Polyphosphate/ATP-NAD Kinase from Yersinia pestis (PDB ID: 4HAO) .

Key structural features likely include:

Detailed crystallographic analysis would involve:

• Expression and purification of high-quality protein

• Crystallization trials with various precipitants and conditions

• X-ray diffraction data collection (typically at 2-3 Å resolution)

• Structure solution by molecular replacement using related NADK structures

• Refinement and validation of the final model

What is the evolutionary significance of phosphoryl donor specificity in NADKs?

The distribution of ATP-specific and poly(P)/ATP-NADKs across bacterial taxa provides insight into the evolution of these enzymes. Poly(P)/ATP-NADKs are found throughout Gram-positive bacteria and Archaea, whereas ATP-specific NADKs are found in Gram-negative α- and γ-proteobacteria (including I. loihiensis) and eukaryotes .

This distribution pattern suggests that:

• Ancestral enzyme type: Poly(P)/ATP-NADKs likely represent the ancestral form of the enzyme, as poly(P) is believed to be an ancient energy currency that predates ATP in evolutionary history .

• Evolutionary transition: NADKs appear to have evolved from poly(P)/ATP-NADKs into ATP-specific NADKs in certain lineages, possibly reflecting adaptation to environments where ATP became the dominant energy currency .

• Convergent evolution: The fact that a single amino acid substitution can switch specificity suggests that this transition may have occurred independently in multiple lineages through convergent evolution .

• Functional significance: The shift from poly(P) utilization to ATP specificity may reflect adaptation to different environmental niches, such as the deep-sea hydrothermal vent environment where I. loihiensis is found .

Understanding this evolutionary trajectory provides insight into both the history of metabolic enzymes and potential strategies for enzyme engineering to recover ancestral functions.

What methodologies can be used to assess the thermal and pH stability of recombinant I. loihiensis ppnK?

Comprehensive assessment of thermal and pH stability requires multiple complementary approaches:

• Activity-based assays:

  • Incubate enzyme samples at different temperatures (4-80°C) for varying time periods

  • Measure residual activity after incubation using standard NADK assay

  • Calculate half-life at each temperature

  • For pH stability, pre-incubate enzyme in buffers of different pH (4-10) before assaying under standard conditions

• Biophysical characterization:

  • Differential scanning calorimetry (DSC) to determine melting temperature (Tm)

  • Circular dichroism (CD) spectroscopy to monitor secondary structure changes with temperature or pH

  • Differential light scattering to detect protein aggregation onset temperature

  • Intrinsic tryptophan fluorescence to monitor tertiary structure changes

• Long-term stability assessment:

  • Store enzyme under different conditions (temperature, pH, buffer components)

  • Periodically measure activity and protein concentration

  • Determine optimal storage conditions and shelf-life

For I. loihiensis enzymes specifically, given the organism's environmental adaptability (4°C to 46°C temperature range and 0.5% to 20% NaCl salinity tolerance), it would be particularly interesting to examine how the ppnK enzyme's stability reflects these adaptations .

How can I design experiments to characterize the metal ion dependence of I. loihiensis ppnK?

NAD kinases typically require divalent metal ions as cofactors. A systematic approach to characterizing metal ion dependence would include:

• Metal requirement determination:

  • Prepare enzyme in metal-free condition using chelating agents (EDTA)

  • Test activity with and without metal ions to confirm requirement

• Metal preference screening:

  • Assay enzyme activity in the presence of various divalent metals (Mg²⁺, Mn²⁺, Co²⁺, Zn²⁺, Ca²⁺, Ni²⁺, Cu²⁺, Fe²⁺)

  • Use standardized concentrations (typically 1-5 mM) for initial screening

  • Compare relative activities to identify preferred metal cofactors

• Dose-response analysis:

  • For metals supporting activity, determine optimal concentration

  • Measure activity across range of metal concentrations (0.1-20 mM)

  • Plot activity versus concentration to identify optimal and inhibitory concentrations

• Kinetic parameter determination with different metals:

  • Determine Km and kcat values with preferred metals

  • Compare catalytic efficiency (kcat/Km) with different metal cofactors

Based on studies with other enzymes from I. loihiensis and related bacteria, it's likely that Mg²⁺ would serve as the primary cofactor, with Mn²⁺ potentially supporting activity as well .

What approaches can be used to study substrate specificity beyond NAD+ for I. loihiensis ppnK?

While NADKs primarily phosphorylate NAD+, some can act on alternative substrates. A comprehensive substrate specificity study would include:

• Substrate screening:

  • Test activity with NAD+ analogs (NADH, deamino-NAD+, nicotinamide mononucleotide)

  • Assess activity with other nucleotides (NDP, NMP compounds)

  • Screen for activity with small molecule substrates containing hydroxyl groups

• Kinetic characterization of alternative substrates:

  • For substrates showing activity, determine Km, Vmax, and kcat

  • Calculate specificity constants (kcat/Km) for comparison with NAD+

  • Construct a specificity profile ranking substrates by catalytic efficiency

• Structural basis for specificity:

  • Use molecular docking to predict binding modes of alternative substrates

  • Identify key residues involved in substrate recognition

  • Design site-directed mutagenesis experiments to alter specificity

• Product confirmation:

  • Use HPLC, mass spectrometry, or NMR to confirm the identity of phosphorylated products

  • Verify regioselectivity of phosphorylation (e.g., 2'-OH vs. 3'-OH)

These experiments would provide a comprehensive understanding of the substrate scope of I. loihiensis ppnK and potentially identify novel activities that could be exploited for biotechnological applications.

How can I optimize expression conditions to maximize yield of active recombinant I. loihiensis ppnK?

Optimizing expression conditions requires a systematic approach to test multiple variables:

• Expression host selection:

  • Test multiple E. coli strains (BL21(DE3), Rosetta, Arctic Express, etc.)

  • Consider other expression hosts for difficult proteins (Pseudomonas, yeast systems)

• Expression vector optimization:

  • Test different promoters (T7, tac, araBAD)

  • Optimize codon usage for E. coli

  • Test various fusion tags (His6, GST, MBP, SUMO) for improved solubility

• Induction parameter optimization:

  • Test range of inducer concentrations (0.01-1.0 mM IPTG)

  • Optimize induction OD600 (early-log to late-log phase)

  • Test various induction temperatures (16°C, 25°C, 30°C, 37°C)

  • Optimize induction duration (4h, 8h, 16h, 24h)

• Media and supplement optimization:

  • Compare rich media (LB, TB, 2YT) and defined media

  • Test addition of osmolytes (sorbitol, betaine)

  • Supplement with cofactors or metal ions if required

  • Consider auto-induction media

• Design of experiments (DoE) approach:

  • Use factorial design to test multiple parameters simultaneously

  • Identify significant parameters and potential interactions

  • Conduct response surface methodology to find optimal conditions

The optimization process should include regular assessment of both protein yield and specific activity to ensure that conditions producing high yields also maintain enzyme functionality .

What are the potential causes and solutions for poor activity of recombinant I. loihiensis ppnK?

Several factors can contribute to poor enzyme activity:

For I. loihiensis ppnK specifically, considering that it comes from a deep-sea bacterium that lives in specialized conditions, attention should be paid to:

• Salt concentration in buffers (I. loihiensis tolerates 0.5-20% NaCl)

• Temperature effects (active range likely 4-46°C)

• Potential requirement for specific metal ions found in its native environment

How can I resolve conflicting data regarding the phosphoryl donor specificity of I. loihiensis ppnK variants?

When faced with conflicting results regarding phosphoryl donor specificity:

• Verify protein integrity:

  • Confirm sequence by mass spectrometry or N-terminal sequencing

  • Assess purity by SDS-PAGE and verify no proteolytic degradation

  • Check for proper folding using circular dichroism

• Standardize assay conditions:

  • Ensure consistent buffer composition across experiments

  • Standardize metal cofactor type and concentration

  • Control temperature and pH precisely

  • Use consistent substrate preparation methods

• Cross-validate with multiple assay methods:

  • Compare direct (product formation) and coupled assay systems

  • Use alternative detection methods (spectrophotometric, HPLC, radiometric)

  • Validate results using different protein batches

• Examine enzyme kinetics in detail:

  • Determine full kinetic parameters (Km, kcat) for both ATP and poly(P)

  • Test for substrate or product inhibition effects

  • Assess potential allosteric regulation

• Consider environmental factors:

  • Test activity across physiologically relevant conditions

  • Examine effects of ionic strength and specific ions

  • Consider temperature effects on specificity

This systematic approach can help resolve discrepancies and provide a more complete understanding of the enzyme's true specificity profile.

What techniques can I use to investigate the structural changes associated with the specificity-determining mutation in I. loihiensis ppnK?

Several complementary techniques can elucidate structural changes:

• X-ray crystallography:

  • Crystallize both wild-type and mutant enzymes

  • Solve structures with bound substrates/analogs when possible

  • Compare active site architecture and substrate binding modes

  • Identify changes in protein dynamics through B-factor analysis

• Solution-based structural techniques:

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to probe conformational differences

  • Small-angle X-ray scattering (SAXS) to assess global conformational changes

  • Nuclear magnetic resonance (NMR) for residue-specific information if protein size permits

• Computational approaches:

  • Molecular dynamics simulations to assess dynamic effects of mutations

  • Molecular docking to predict changes in substrate binding

  • Free energy calculations to quantify changes in binding affinity

• Biophysical characterization:

  • Circular dichroism spectroscopy to examine secondary structure changes

  • Fluorescence spectroscopy to probe tertiary structure alterations

  • Differential scanning calorimetry to assess stability differences

• Functional probes:

  • Chemical cross-linking followed by mass spectrometry

  • Site-directed spin labeling for electron paramagnetic resonance (EPR)

  • Fluorescence resonance energy transfer (FRET) to measure distances between labeled residues

These approaches would provide multilevel insights into how a single amino acid substitution can dramatically alter substrate specificity.

How can engineered I. loihiensis ppnK variants be utilized in synthetic biology applications?

Engineered variants of I. loihiensis ppnK with modified specificity could find several applications in synthetic biology:

• NADPH regeneration systems:

  • Design regeneration cycles for NADPH-dependent biocatalysis

  • Create polycistronic expression systems coupling NADK with NADP+-dependent enzymes

  • Engineer metabolic pathways with improved NADPH availability for product synthesis

• Biosensors and reporting systems:

  • Develop biosensors for polyphosphate levels in environmental samples

  • Create coupled enzyme systems for detection of NAD+ or ATP

  • Design fluorescent or colorimetric assays based on NADP+ production

• Metabolic engineering applications:

  • Enhance NADPH availability for production of valuable metabolites

  • Control cellular redox balance in engineered microorganisms

  • Build synthetic pathways utilizing polyphosphate as an energy source

• Structural biology platforms:

  • Use as a model system for studying phosphoryl transfer mechanisms

  • Develop platforms for screening enzyme evolution

  • Create systems for studying enzyme adaptation to extreme environments

For these applications, the deep-sea origin of I. loihiensis ppnK may provide unique advantages in terms of stability and adaptability to various conditions .

What are the most promising directions for further engineering of I. loihiensis ppnK?

Several promising engineering directions include:

• Enhancing catalytic efficiency:

  • Structure-guided mutagenesis of active site residues

  • Directed evolution to improve kcat/Km values

  • Engineering substrate binding pocket for improved affinity

• Expanding substrate scope:

  • Engineer variants capable of phosphorylating alternative substrates

  • Develop enzymes with broader nucleotide specificity

  • Create variants with novel regioselectivity

• Tailoring stability for specific applications:

  • Engineer thermostable variants for high-temperature processes

  • Develop cold-active variants for low-temperature applications

  • Create pH-tolerant variants for diverse reaction conditions

• Altering regulatory properties:

  • Remove feedback inhibition mechanisms

  • Engineer allosteric regulation responses

  • Develop variants with altered metal cofactor requirements

• Creating fusion proteins:

  • Design bifunctional enzymes coupling NADK with related metabolic enzymes

  • Create immobilization-ready variants with appropriate tags

  • Develop cellular targeting variants for in vivo applications

These engineering efforts could significantly expand the utility of I. loihiensis ppnK in both research and biotechnological applications.