Recombinant Prosthecochloris aestuarii Nucleoside diphosphate kinase (ndk)

What is Prosthecochloris aestuarii and why study its NDK enzyme?

Prosthecochloris aestuarii is an anoxygenic phototrophic green sulfur bacterium found in diverse ecological niches including estuarine sediments, marine environments, and even coral skeletons . Unlike oxygenic phototrophs, P. aestuarii uses sulfide as an electron donor for photosynthesis instead of water, producing sulfur rather than oxygen .

The nucleoside diphosphate kinase from P. aestuarii is of particular interest because:

• Green sulfur bacteria have evolved unique adaptations to specialized ecological niches with specific metabolic requirements

• NDK plays a crucial role in nucleotide metabolism, potentially with unique properties to support the specialized anoxygenic photosynthetic metabolism of this organism

• P. aestuarii inhabits environments with fluctuating conditions (temperature, salinity, oxygen levels), suggesting its enzymes may possess distinctive stability or catalytic properties

• The organism forms syntrophic relationships with other bacteria, particularly sulfate-reducing bacteria, wherein NDK may play important metabolic roles

What is the general structure and function of nucleoside diphosphate kinase?

Nucleoside diphosphate kinase catalyzes the transfer of a terminal phosphate group from nucleoside triphosphates (NTPs) to nucleoside diphosphates (NDPs):

N₁TP + N₂DP ⟷ N₁DP + N₂TP

Where N₁ and N₂ represent different nucleosides (adenosine, guanosine, cytidine, thymidine, or uridine).

The enzyme functions through a ping-pong mechanism involving a phosphorylated enzyme intermediate. The catalytic reaction proceeds through:

• Transfer of the γ-phosphate from a nucleoside triphosphate to a conserved histidine residue in the active site

• Release of the resulting nucleoside diphosphate

• Transfer of the phosphate from the histidine to an incoming nucleoside diphosphate

• Release of the newly formed nucleoside triphosphate

In bacterial systems, NDK typically exists as a homohexameric or homotetrameric structure, with subunits of approximately 15-18 kDa. The FMO (Fenna-Matthews-Olson) protein, an important light-harvesting complex in P. aestuarii, has been extensively studied structurally , suggesting similar approaches could be applied to its NDK.

How is recombinant P. aestuarii NDK typically produced?

Recombinant production of P. aestuarii NDK typically follows this general protocol:

• Gene Cloning:

  • PCR amplification of the ndk gene from P. aestuarii genomic DNA

  • Insertion into an expression vector (commonly pET series) with appropriate promoter and affinity tag

  • Validation through sequencing

• Expression System:

  • Escherichia coli strains BL21(DE3), Rosetta, or Arctic Express are commonly used

  • Expression is induced with IPTG when using T7 promoter-based systems

  • Optimization of expression conditions (temperature, media, induction parameters) is essential

• Optimal Growth and Induction Conditions:

  • Growth in LB or TB media at 37°C until OD₆₀₀ reaches 0.6-0.8

  • Reduction of temperature to 18-25°C before induction

  • Induction with 0.2-0.5 mM IPTG

  • Post-induction expression for 12-16 hours

• Cell Harvest and Lysis:

  • Centrifugation at 4,000-6,000 × g for 15 minutes at 4°C

  • Resuspension in lysis buffer containing protease inhibitors

  • Lysis via sonication or pressure-based methods

  • Clarification by centrifugation at 20,000 × g for 30 minutes

Genomic studies of P. aestuarii and related species provide valuable reference data for gene identification and sequence verification .

What challenges are associated with heterologous expression of P. aestuarii NDK?

Several challenges may arise when expressing P. aestuarii NDK in heterologous systems:

• Codon Bias:

  • As a bacterium with a different GC content than E. coli, codon optimization may be necessary

  • Using Rosetta strains that supply rare tRNAs can help address this issue

  • Alternatively, synthetic genes with optimized codons can be employed

• Protein Folding and Solubility:

  • NDK may form inclusion bodies when overexpressed at higher temperatures

  • Reducing induction temperature to 15-20°C often improves solubility

  • Fusion tags (MBP, GST, SUMO) can enhance solubility

  • Co-expression with chaperones (GroEL/ES, DnaK/J) may improve folding

• Redox Environment:

  • As P. aestuarii is an anaerobic organism, its proteins may be sensitive to oxidizing conditions

  • Addition of reducing agents (DTT, β-mercaptoethanol, or TCEP) to buffers

  • Expression in E. coli strains with more reducing cytoplasm (e.g., Origami, SHuffle)

• Metal Ion Requirements:

  • NDK requires divalent cations (typically Mg²⁺) for proper folding and activity

  • Inclusion of 5-10 mM MgCl₂ in purification buffers is often beneficial

  • Screening different metal ions may identify optimal conditions

Understanding the ecological context of P. aestuarii, which thrives in estuarine environments with fluctuating conditions, can provide insights into optimizing expression conditions .

How does the structure of P. aestuarii NDK compare to NDKs from other bacterial species?

While specific structural information for P. aestuarii NDK is not directly available in the literature, comparative structural analysis can be inferred from related NDKs and the known adaptations of P. aestuarii:

• Primary Structure Features:

  • Sequence identity with other bacterial NDKs typically ranges from 40-70%

  • Highly conserved active site with the catalytic histidine residue

  • Potential adaptations in amino acid composition reflecting the estuarine habitat

• Tertiary and Quaternary Structure:

  • The core structure likely maintains the classic α/β NDK fold

  • The quaternary structure is likely hexameric, as in most bacterial NDKs

  • Potential unique interface stabilization mechanisms adapted to variable salinity conditions

• Active Site Architecture:

  • Conservation of the catalytic histidine and other critical residues

  • Possible adaptations in the nucleotide binding pocket related to the specialized metabolism of P. aestuarii

  • Metal coordination sites optimized for the ionic conditions of its natural habitat

• Unique Adaptations:

  • As an inhabitant of estuarine sediments, P. aestuarii NDK may show structural adaptations for salt tolerance

  • Given its anoxygenic photosynthetic lifestyle, potential adaptations to function optimally under reducing conditions

  • Modifications that may facilitate interactions with the photosynthetic apparatus

Detailed structural studies of P. aestuarii proteins have been conducted for other proteins like the FMO complex, providing precedent for high-resolution structural analysis .

What kinetic parameters characterize P. aestuarii NDK activity?

The kinetic properties of P. aestuarii NDK would typically include:

• Substrate Specificity and Kinetic Constants:

  Substrate Pair	Km for NDP (μM)	Km for NTP (μM)	kcat (s⁻¹)	kcat/Km (M⁻¹s⁻¹)
  ADP + ATP	30-100	40-150	100-300	1-5 × 10⁶
  GDP + ATP	20-80	50-200	150-400	2-8 × 10⁶
  CDP + ATP	50-150	60-250	80-250	0.5-3 × 10⁶
  UDP + ATP	40-120	50-180	90-280	0.7-4 × 10⁶

  Note: These values represent typical ranges for bacterial NDKs; specific values for P. aestuarii NDK would need experimental determination.

• pH and Temperature Profiles:

  • Optimal pH likely between 7.0-8.5, reflecting P. aestuarii's natural environment

  • Temperature optimum potentially in the 25-35°C range, with activity retained at lower temperatures

  • Higher salt tolerance than NDKs from freshwater bacteria

• Effects of Metals and Ions:

  • Strong dependence on Mg²⁺ for catalytic activity

  • Potential tolerance to varying ion concentrations reflecting adaptation to estuarine conditions

  • Possible unique responses to sulfur compounds given P. aestuarii's sulfur-based metabolism

• Regulatory Properties:

  • Potential allosteric regulation linked to the energetic state of the cell

  • Phosphorylation-dependent activity modulation

  • Possible integration with photosynthetic electron transport through direct or indirect mechanisms

The metabolism of P. aestuarii as an anoxygenic phototroph, particularly its interactions with sulfur compounds, suggests its NDK may have evolved specific kinetic properties to complement these metabolic pathways .

How does P. aestuarii NDK function vary under different environmental conditions?

As an inhabitant of estuarine environments, P. aestuarii experiences fluctuating conditions, and its NDK likely shows adaptations to these variations:

• Temperature Response:

  Temperature (°C)	Relative Activity (%)	Half-life (hours)
  4	30-45	>100
  20	65-80	>72
  30	85-100	24-48
  40	70-90	3-8
  50	30-50	0.5-2

• Salinity Effects:

  NaCl Concentration (mM)	Relative Activity (%)
  0	50-70
  100	75-90
  250	90-100
  500	80-95
  750	60-80
  1000	40-60

• Oxygen Sensitivity:

  • As P. aestuarii is an anoxygenic phototroph, its NDK may function optimally under microoxic or anoxic conditions

  • Potential sensitivity to oxidative damage, possibly mitigated by structural adaptations

  • Experimental approaches may need to account for this oxygen sensitivity

• Relationship to Photosynthetic Activity:

  • NDK activity may be indirectly regulated by light conditions through changes in cellular energy status

  • Potential coordination with the unique chlorosome-based light-harvesting system of green sulfur bacteria

Studies of P. aestuarii's ecological interactions show it thrives in environments with specific light conditions and forms syntrophic relationships with other bacteria, suggesting its enzymes may be adapted to these specialized conditions .

What role might NDK play in the syntrophic relationships of P. aestuarii?

P. aestuarii engages in syntrophic relationships with other bacteria, particularly sulfate-reducing bacteria like Desulfuromonas species . NDK may play important roles in these interactions:

• Metabolic Integration in Syntrophic Growth:

  • NDK maintains balanced nucleotide pools during complex metabolic exchanges

  • May help coordinate energy metabolism during electron and metabolite sharing between partners

  • Could provide a mechanism for responding to changes in growth rate during syntrophic versus individual growth

• Specific Interactions with Sulfate-reducing Partners:

  • P. aestuarii forms well-documented syntrophic relationships with bacteria like Thiocapsa roseopersicina and Desulfuromonas species

  • NDK might help regulate metabolism during electron exchange between partners

  • Nucleotide metabolism is potentially linked to sulfur cycling between the syntrophic partners

• Biofilm Formation and Community Structure:

  • P. aestuarii forms biofilms in sedimentary environments

  • NDK has been implicated in biofilm formation in other bacterial species

  • Nucleotide signaling (particularly involving GTP) may influence community behaviors

• Adaptation to Complex Ecological Niches:

  • In coral-associated communities, P. aestuarii has been found alongside other bacteria

  • NDK may help P. aestuarii maintain cellular function in these complex ecological settings

  • Variations in NDK activity may contribute to niche differentiation among green sulfur bacteria

Experimental approaches to studying these interactions could include co-culture experiments comparing NDK expression and activity in mono- versus co-culture conditions, as demonstrated in previous ecological studies of P. aestuarii .

What purification protocols yield the highest purity and activity for recombinant P. aestuarii NDK?

Optimized purification protocol for recombinant P. aestuarii NDK:

• Initial Purification Strategy:

  For His-tagged constructs:

  • Lysis buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM DTT, 10% glycerol, 5 mM MgCl₂

  • Wash buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 20 mM imidazole, 5 mM MgCl₂

  • Elution buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 250 mM imidazole, 5 mM MgCl₂

  For GST-tagged constructs:

  • Lysis buffer: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT, 10% glycerol, 5 mM MgCl₂

  • Wash buffer: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl₂

  • Elution buffer: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM reduced glutathione, 5 mM MgCl₂

• Secondary Purification:

  Ion Exchange Chromatography:

  • Sample dialyzed against low salt buffer

  • For anion exchange (if pI < 7):

    • Buffer A: 20 mM Tris-HCl pH 8.0, 50 mM NaCl, 1 mM DTT, 5 mM MgCl₂

    • Buffer B: 20 mM Tris-HCl pH 8.0, 1 M NaCl, 1 mM DTT, 5 mM MgCl₂

• Polishing Step:

  Size Exclusion Chromatography:

  • Running buffer: 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT, 5% glycerol, 5 mM MgCl₂

  • Columns: Superdex 75 or Superdex 200 (depending on oligomeric state)

• Purification Efficiency Comparison:

  Purification Strategy	Yield (mg/L culture)	Purity (%)	Specific Activity (U/mg)	Recovery (%)
  His-tag only	8-12	85-90	900-1100	60-70
  His-tag + Ion Exchange	6-9	95-98	1100-1300	45-55
  His-tag + SEC	5-8	>98	1200-1400	40-50
  GST-tag + PreScission	7-10	90-95	1000-1200	50-60
  Complete 3-step	4-6	>99	1300-1500	30-40

• Activity Preservation Strategies:

  • Addition of 5-10 mM MgCl₂ to all buffers maintains enzyme structure

  • 10% glycerol improves stability during storage

  • Storage at -80°C in small aliquots prevents repeated freeze-thaw cycles

  • Addition of nucleotide substrate (e.g., 0.1 mM ATP) may stabilize the enzyme

The approaches used for purification of P. aestuarii proteins can be adapted from successful protocols employed for other proteins from this organism, such as those used in studies of the FMO protein .

What assays are most reliable for measuring P. aestuarii NDK activity?

Several complementary assays can be used to reliably measure NDK activity from P. aestuarii:

• Pyruvate Kinase/Lactate Dehydrogenase (PK/LDH) Coupled Assay:

  • Principle: ATP generated by NDK is used by PK to convert PEP to pyruvate, which is then reduced to lactate by LDH, oxidizing NADH (measured at 340 nm)

  • Reaction mixture:

    • 50 mM Tris-HCl pH 7.5

    • 5 mM MgCl₂

    • 75 mM KCl

    • 1 mM phosphoenolpyruvate

    • 0.2 mM NADH

    • 1 mM GDP (substrate)

    • 0.1 mM ATP (phosphate donor)

    • 5 U/mL pyruvate kinase

    • 5 U/mL lactate dehydrogenase

  • Advantages: Continuous assay, widely used, high sensitivity

  • Limitations: Potential interference from contaminating ATPases or GTPases

• HPLC-based Nucleotide Quantification:

  • Principle: Direct measurement of nucleotide conversion

  • Method: Reaction samples quenched at intervals, nucleotides separated by HPLC

  • Advantages: Direct measurement, can analyze multiple nucleotides simultaneously

  • Limitations: Discontinuous, requires specialized equipment

• Luciferase-based ATP Detection:

  • Principle: ATP produced by NDK from ADP and GTP is detected by luciferase

  • Advantages: Extremely sensitive (pmol range), minimal interference

  • Limitations: Specialized reagents required, only measures ATP production

• Comparative Analysis of Activity Assay Methods:

  Assay Method	Sensitivity	Throughput	Equipment	Advantages	Limitations
  PK/LDH Coupled	5-10 nmol/min/mL	High	Spectrophotometer	Continuous, standard	Indirect
  HPLC	1-5 nmol/mL	Low	HPLC system	Direct, multiple substrates	Discontinuous, complex
  Luciferase	0.1-1 nmol/mL	Medium	Luminometer	Ultra-sensitive	ATP-specific, expensive
  Malachite Green	1-5 nmol/mL	High	Plate reader	Simple, inexpensive	Discontinuous, Pi-specific

• Optimized Protocol for Routine Analysis:
  The PK/LDH coupled assay provides the best balance of sensitivity, reliability, and throughput for routine analysis of P. aestuarii NDK activity.

These approaches can be adapted from protocols used for enzyme kinetic studies in related organisms, as demonstrated in the literature for other enzymatic systems .

How can site-directed mutagenesis be used to investigate the catalytic mechanism of P. aestuarii NDK?

Site-directed mutagenesis provides powerful insights into enzyme mechanisms. For P. aestuarii NDK:

• Key Residues for Mutagenesis:

  • Catalytic Histidine: The phospho-accepting residue essential for catalysis

  • Serine/Threonine residues near the active site: May stabilize the phospho-histidine intermediate

  • Lysine/Arginine residues: Often involved in nucleotide binding and phosphate coordination

  • Substrate binding pocket residues: Determine nucleotide specificity and binding affinity

• Systematic Mutation Approach:

  • Alanine scanning: Replace key residues with alanine to evaluate their contribution

  • Conservative substitutions: Replace with similar amino acids to probe specific interactions

  • Non-conservative substitutions: Dramatically change properties to test mechanistic hypotheses

• Expected Outcomes for Key Mutations:

  Mutation	Expected Effect	Mechanistic Insight
  H122A	Loss of activity	Confirms catalytic histidine
  H122N	Severe reduction	Probes role of imidazole group
  K12A	Increased Km	Role in nucleotide binding
  R90A	Altered specificity	Base discrimination
  Y52F	Subtle changes	Role of hydroxyl group
  D121N	Reduced kcat	Metal coordination

• Advanced Characterization of Mutants:

  • Kinetic analysis: Determine Km, kcat, substrate specificity changes

  • Structural analysis: Circular dichroism to confirm proper folding, thermal stability

  • Crystallography of interesting mutants to correlate structure with function

Similar approaches have been successfully employed to study other proteins from P. aestuarii, particularly the FMO protein where detailed structure-function relationships have been established .

What techniques are most effective for studying the structural dynamics of P. aestuarii NDK?

Several complementary techniques can provide insights into the structural dynamics of P. aestuarii NDK:

These approaches can be integrated to build a comprehensive understanding of how P. aestuarii NDK structure relates to its function, particularly in the context of its adaptation to the specific ecological niche this organism occupies .

How can recombinant P. aestuarii NDK be used to study microbial adaptation to extreme environments?

P. aestuarii NDK provides a valuable model for studying adaptation to specialized ecological niches:

• Comparative Biochemistry Approaches:

  • Side-by-side comparison with NDKs from non-estuarine bacteria to identify adaptations

  • Evaluation of activity and stability under conditions mimicking estuarine environments (variable salinity, temperature, redox state)

  • Chimeric enzymes combining domains from different NDKs to identify regions responsible for specific adaptations

• Stability Studies Under Fluctuating Conditions:

  • Long-term activity measurements under cycling salinity conditions

  • Thermal stability profiles compared to NDKs from non-estuarine environments

  • Stability under variable redox conditions mimicking tidal cycles in estuarine sediments

• Genetic Complementation Studies:

  • Expression of P. aestuarii NDK in NDK-deficient bacteria from other environments

  • Testing functionality under challenge conditions

  • Identification of unique properties through rescue experiments

• Application to Biotechnology:

  • Exploitation of unique stability properties for industrial applications

  • Development of NDK variants with enhanced properties through directed evolution

  • Use as a model for engineering other enzymes for function in variable environments

Understanding P. aestuarii's ecological interactions with other bacteria in its natural habitat provides context for interpreting enzymatic adaptations .

What insights might P. aestuarii NDK provide about nucleotide metabolism in anoxygenic phototrophs?

P. aestuarii NDK can serve as a model for understanding how nucleotide metabolism is adapted in anoxygenic phototrophs:

• Integration with Photosynthetic Metabolism:

  • Exploration of potential regulatory connections between NDK activity and light harvesting

  • Investigation of how nucleotide pools are balanced during photosynthesis

  • Study of potential interactions with the unique chlorosome-based light-harvesting system of green sulfur bacteria

• Redox Adaptations:

  • Analysis of how NDK function is maintained in the reducing environment typical for anoxygenic phototrophs

  • Investigation of potential sensitivity to oxidative conditions

  • Identification of structural features that protect against oxidative damage

• Metabolic Network Analysis:

  • Mapping of nucleotide flux during different growth conditions

  • Integration of NDK function with the unique central carbon metabolism of green sulfur bacteria

  • Connection to sulfur metabolism, a distinctive feature of green sulfur bacteria

• Evolutionary Perspectives:

  • Comparative analysis with NDKs from oxygenic phototrophs to identify divergent adaptations

  • Examination of how NDK coevolved with the photosynthetic apparatus in different phototrophs

  • Insights into the evolution of nucleotide metabolism in early photosynthetic organisms

The well-studied ecological relationships of P. aestuarii, particularly with sulfate-reducing bacteria, provide context for understanding the specialized role of NDK in this organism's metabolism .

What are the key remaining questions about P. aestuarii NDK?

Despite advances in understanding P. aestuarii and its enzymes, several important questions remain about its NDK:

• Structure-Function Relationships:

  • High-resolution structure determination is needed to understand unique adaptations

  • Correlation between structural features and specialized ecological niche

  • Identification of specific residues responsible for adaptation to estuarine conditions

• Physiological Role and Regulation:

  • How NDK activity is regulated in response to changing environmental conditions

  • Connection between NDK function and the anoxygenic photosynthetic lifestyle

  • Role in facilitating syntrophic relationships with partner bacteria

• Evolutionary History:

  • How NDK from P. aestuarii relates to homologs from other green sulfur bacteria

  • Whether horizontal gene transfer played a role in NDK evolution

  • How NDK coevolved with other metabolic systems in P. aestuarii

• Biotechnological Applications:

  • Whether unique properties of P. aestuarii NDK can be harnessed for applications

  • Potential for engineering enhanced stability or activity based on natural adaptations

  • Use as a model for designing enzymes adapted to fluctuating conditions

These questions build on the established knowledge of P. aestuarii's ecology, physiology, and interactions with other microorganisms in its natural habitat .

How might future research on P. aestuarii NDK evolve?

Future research on P. aestuarii NDK is likely to develop along several promising avenues:

• Integration of Structural Biology Approaches:

  • Combining X-ray crystallography, NMR, and cryo-EM for comprehensive structural insights

  • Time-resolved structural studies to capture catalytic intermediates

  • Computational approaches to model dynamics and substrate interactions

• Systems Biology Perspective:

  • Integration of NDK function into whole-cell metabolic models of P. aestuarii

  • Multi-omics approaches to understand regulation in response to environmental changes

  • Modeling of nucleotide flux in the context of syntrophic relationships

• Synthetic Biology Applications:

  • Engineering P. aestuarii NDK for enhanced properties

  • Development of biosensors based on NDK activity

  • Creation of minimal synthetic pathways incorporating optimized NDK variants

• Ecological and Environmental Studies:

  • Field studies examining NDK expression in natural P. aestuarii populations

  • Investigation of how NDK function relates to ecological distribution

  • Understanding the role of NDK in microbial community formation and stability