Recombinant Gloeobacter violaceus Acetate kinase (ackA)

What is the function of acetate kinase (ackA) in Gloeobacter violaceus?

Acetate kinase (ackA) in Gloeobacter violaceus is a crucial enzyme that catalyzes substrate-level phosphorylation in the phosphotrans-acetylase-acetate kinase (Pta-Ack) pathway. This enzyme specifically catalyzes the reversible conversion of acetyl phosphate to acetate while generating ATP, serving as a vital secondary energy-yielding pathway. In cyanobacteria like G. violaceus, ackA plays a significant role in energy metabolism, particularly during the stationary phase when the organism utilizes accumulated acetate as a carbon source. The enzyme functions by transferring a phosphate group from acetyl phosphate to ADP, producing ATP and acetate, which is essential for bacterial survival under nutrient-limited or hostile environmental conditions .

How does Gloeobacter violaceus ackA differ structurally from acetate kinases in other bacteria?

Gloeobacter violaceus ackA exhibits unique structural features that distinguish it from acetate kinases in other bacterial species. Comparative structural analyses with acetate kinases from other bacteria typically reveal notable variations in the amino acid sequences and tertiary structures. Similar to the differences observed between S. aureus ackA and the enzymes from Mycobacterium avium and Salmonella typhimurium (with RMSD values of 1.877 Å and 2.141 Å respectively), G. violaceus ackA likely possesses distinctive structural elements .

The G. violaceus ackA structure contains conserved domains including the ACKA-1 and ACKA-2 domains, with an ATP binding site typically present within the ACKA-2 domain. Additionally, it may contain unique serine/threonine phosphorylation sites that regulate enzyme function. These structural differences explain why acetate kinase functions are differently placed across bacterial species and may contribute to G. violaceus's ability to survive in its distinctive habitat as one of the most primitive cyanobacteria lacking thylakoid membranes .

What are the typical kinetic parameters of recombinant Gloeobacter violaceus ackA?

Recombinant G. violaceus acetate kinase typically exhibits specific enzymatic properties that can be characterized through kinetic analysis. Based on comparative data with other bacterial acetate kinases, the enzyme would likely demonstrate the following parameters:

These kinetic parameters would typically be determined using the coupled assay system with pyruvate kinase and lactate dehydrogenase, monitoring the decrease in NADH absorbance at 340 nm. The precise values might vary depending on the specific recombinant expression system and purification methods employed .

What are the optimal conditions for recombinant expression of Gloeobacter violaceus ackA?

The optimal conditions for recombinant expression of G. violaceus ackA involve careful selection of expression systems and fine-tuning of expression parameters. A methodical approach includes:

• Expression System Selection: E. coli expression systems using pET vectors or pQE vectors (similar to the pQE 30 used for S. aureus ackA) are recommended. BL21(DE3) or DH5α strains typically yield good results for cyanobacterial proteins.

• Vector Design: The gene should be cloned with appropriate restriction sites (e.g., SalI and HindIII) and optimally include a His-tag for purification. Codon optimization for E. coli expression may be necessary given the GC-rich content of cyanobacterial genes.

• Culture Conditions:

  • Growth medium: LB or 2xYT supplemented with appropriate antibiotics

  • Temperature: Initial growth at 37°C until OD600 reaches 0.6-0.8

  • Post-induction temperature: 25-30°C (lower temperatures often improve solubility)

  • Induction: 0.5-1.0 mM IPTG, typically for 5-16 hours

• Optimization Parameters:

  • IPTG concentration can be titrated between 0.1-1.0 mM

  • Post-induction incubation time can be optimized between 4-24 hours

  • Addition of 5-10% glycerol to the culture medium can improve protein solubility

  • Supplementation with 0.2-0.5% glucose can reduce basal expression before induction

The PCR conditions for amplifying the ackA gene would typically involve initial denaturation at 94°C for 5 minutes, followed by 35 cycles of denaturation (94°C, 50 seconds), annealing (optimized between 40-55°C, 45 seconds), and extension (72°C, 2 minutes), with a final extension at 72°C for 10 minutes .

What purification strategy yields the highest purity and activity for recombinant G. violaceus ackA?

A multi-step purification strategy is recommended to obtain high-purity, active recombinant G. violaceus ackA:

• Cell Lysis: Cells should be harvested by centrifugation (6,000×g, 15 minutes, 4°C) and resuspended in lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM PMSF, 1 mg/ml lysozyme). After incubation on ice for 30 minutes, sonication should be performed (6-10 cycles of 30 seconds on/30 seconds off) until the solution becomes clear.

• Initial Purification: For His-tagged proteins, nickel-metal chelate affinity chromatography is the primary purification method:

  • Equilibration buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole

  • Wash buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 20-40 mM imidazole

  • Elution buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, gradient of 50-300 mM imidazole

• Secondary Purification: Size exclusion chromatography (Superdex 200) to remove aggregates and impurities:

  • Running buffer: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol

• Activity Preservation Steps:

  • Addition of 10% glycerol to all buffers improves enzyme stability

  • Including 1 mM DTT can prevent oxidation of cysteine residues

  • Storage buffer should contain 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 20% glycerol, 1 mM DTT

• Quality Control:

  • Purity assessment: SDS-PAGE (10%) should show a single band at approximately 44 kDa

  • Western blot confirmation using anti-His antibodies

  • Activity verification using the coupled enzyme assay system

This purification strategy typically yields protein with >95% purity and specific activity of 3.0-3.5 μM NADH/ml/min, similar to what has been observed with recombinant S. aureus ackA .

How can enzyme activity be accurately measured for recombinant G. violaceus ackA?

Accurate measurement of G. violaceus ackA enzyme activity requires a well-established coupled enzyme assay system. The recommended protocol includes:

• Principle: The assay couples the production of ADP from the acetate kinase reaction to the oxidation of NADH through pyruvate kinase and lactate dehydrogenase reactions. The decrease in NADH absorbance at 340 nm is proportional to acetate kinase activity.

• Reaction Mixture (0.5 ml total volume):

  • 100 mM Tris-HCl buffer (pH 7.3)

  • 2 mM potassium acetate

  • 1.5 mM ATP

  • 2 mM MgCl₂

  • 2 mM phosphoenolpyruvate

  • 0.4 mM NADH

  • 5 units of pyruvate kinase (PK)

  • 10 units of lactate dehydrogenase (LDH)

• Procedure:

  • Prepare all reagents fresh and maintain at 25°C

  • Establish baseline by measuring absorbance without enzyme

  • Add purified recombinant ackA (2-10 μg) or cytosolic fraction

  • Monitor decrease in absorbance at 340 nm for 5 minutes

  • Calculate activity using the extinction coefficient of NADH (6,220 M⁻¹cm⁻¹)

• Data Analysis:

  • Calculate the slope of the linear portion of the curve (ΔA340/min)

  • Convert to μM NADH/min using the formula: Activity = (ΔA340/min) × (reaction volume in ml) / (6.22 × enzyme volume in ml)

  • Express specific activity as μM NADH/min/mg protein

• Controls and Validations:

  • Negative control: Complete reaction mixture without enzyme

  • Positive control: Commercial acetate kinase with known activity

  • Substrate specificity: Replace acetate with propionate or butyrate

This assay method provides reliable and reproducible measurements of acetate kinase activity, allowing for accurate characterization of the recombinant enzyme .

What computational methods are most effective for analyzing the structure-function relationship of G. violaceus ackA?

A comprehensive computational approach to analyzing the structure-function relationship of G. violaceus ackA should incorporate multiple methods:

• Homology Modeling and Structural Prediction:

  • SWISS-MODEL server can be used to build a reliable 3D model based on known acetate kinase structures, such as those from M. avium (PDB: 3P4I) and S. typhimurium (PDB: 3SLC)

  • AlphaFold2 provides highly accurate structure predictions, especially valuable for G. violaceus ackA which may have unique structural elements

  • Model validation using PROCHECK, VERIFY3D, and ERRAT to ensure stereochemical quality

• Comparative Structural Analysis:

  • Root Mean Square Deviation (RMSD) calculations to compare G. violaceus ackA with other known acetate kinase structures

  • Analysis of conserved domains including ACKA-1 (residues 5-16) and ACKA-2 (residues 215-222)

  • Identification of ATP binding site and catalytic residues using molecular docking approaches

• Functional Domain Analysis:

  • Multiple sequence alignment using MUSCLE or Clustal Omega to identify conserved residues

  • Analysis of serine/threonine phosphorylation sites using NetPhos or PhosphoSitePlus

  • Protein-protein interaction prediction using STRING database

• Molecular Dynamics Simulations:

  • GROMACS or NAMD simulations (10-100 ns) to analyze conformational dynamics

  • Analysis of substrate binding and catalytic mechanisms

  • Evaluation of structural stability under various environmental conditions

• Evolutionary Analysis:

  • Phylogenetic analysis using Maximum Likelihood methods

  • Analysis of selective pressure on different domains using PAML

  • Identification of co-evolving residues using mutual information analysis

Implementation of these computational methods would provide valuable insights into the unique structural features of G. violaceus ackA, potentially revealing how its structure influences its enzymatic function and specificity .

How do mutations in key residues affect the catalytic activity of G. violaceus ackA?

Mutations in key residues of G. violaceus ackA can have profound effects on its catalytic activity. A systematic approach to understanding these effects involves:

• Critical Residues for Investigation:

  • ATP binding site residues within the ACKA-2 domain

  • Conserved residues in the ACKA-1 domain responsible for acetate binding

  • Serine/threonine phosphorylation sites that regulate enzyme activity

  • Interface residues involved in potential dimerization

• Common Mutation Effects:

• Specific Mutation Approaches:

  • Alanine scanning mutagenesis to systematically neutralize each potential critical residue

  • Conservative substitutions (e.g., Asp→Glu) to test the importance of specific chemical properties

  • Non-conservative substitutions to dramatically alter local environments

• Experimental Validation Methods:

  • Site-directed mutagenesis using overlap extension PCR

  • Expression and purification of mutant proteins following standard protocols

  • Enzyme kinetic analysis to determine changes in Km, kcat, and catalytic efficiency

  • Thermal stability assessment using differential scanning fluorimetry

  • Structural characterization of mutants by circular dichroism and limited proteolysis

• Structure-Function Correlation:

  • Molecular dynamics simulations of mutants to understand conformational changes

  • Correlation of activity data with structural alterations

  • Mapping of mutation effects onto the 3D structure

These approaches would illuminate the catalytic mechanism of G. violaceus ackA and potentially identify unique features that could be targeted for inhibitor design or enzymatic optimization .

What are the common challenges in expressing soluble recombinant G. violaceus ackA and how can they be addressed?

Researchers frequently encounter several challenges when expressing soluble recombinant G. violaceus ackA. These challenges and their solutions include:

• Inclusion Body Formation:

  • Problem: G. violaceus proteins often form inclusion bodies due to their cyanobacterial origin and potential membrane association.

  • Solutions:

    • Lower induction temperature to 16-20°C

    • Reduce IPTG concentration to 0.1-0.3 mM

    • Co-express with chaperones (GroEL/GroES, DnaK/DnaJ/GrpE)

    • Use solubility-enhancing fusion tags (SUMO, MBP, or TrxA)

    • Add 5-10% glycerol and 0.1-0.5% Triton X-100 to lysis buffer

• Low Expression Yields:

  • Problem: Cyanobacterial genes often contain rare codons for E. coli.

  • Solutions:

    • Use codon-optimized synthetic gene

    • Express in Rosetta or CodonPlus strains

    • Optimize Shine-Dalgarno sequence and spacing

    • Test multiple promoter systems (T7, tac, or ara)

    • Optimize cell density at induction (OD600 of 0.4-0.8)

• Protein Instability:

  • Problem: Recombinant G. violaceus ackA may be unstable during expression or purification.

  • Solutions:

    • Add protease inhibitors (PMSF, EDTA, leupeptin) to all buffers

    • Include stabilizing agents (glycerol, trehalose, or arginine)

    • Maintain strict temperature control during purification (4°C)

    • Avoid freeze-thaw cycles; use single-use aliquots

    • Test different buffer systems (HEPES, Tris, or phosphate)

• Low Enzymatic Activity:

  • Problem: Purified protein shows reduced or no enzymatic activity.

  • Solutions:

    • Add cofactors during purification (Mg²⁺, K⁺)

    • Include reducing agents (DTT or β-mercaptoethanol)

    • Test protein refolding from inclusion bodies if necessary

    • Ensure proper pH during purification and storage (typically pH 7.0-7.5)

    • Verify proper oligomeric state using size exclusion chromatography

• Aggregation During Storage:

  • Problem: Purified protein aggregates during storage.

  • Solutions:

    • Store at higher concentration (>1 mg/ml)

    • Add 20-30% glycerol to storage buffer

    • Test additives like arginine (50-100 mM) or sucrose (5-10%)

    • Determine optimal protein concentration for stability

    • Flash-freeze in liquid nitrogen and store at -80°C

Implementation of these strategies, often in combination, can significantly improve the yield and quality of recombinant G. violaceus ackA for subsequent biochemical and structural studies .

How can enzyme stability be improved for long-term storage and experimental use?

Improving the stability of recombinant G. violaceus acetate kinase for long-term storage and experimental applications requires a systematic approach addressing multiple aspects of protein stability:

• Optimal Buffer Composition:

  • Base buffer: 50 mM Tris-HCl or HEPES at pH 7.2-7.5

  • Salt: 100-150 mM NaCl or KCl to maintain ionic strength

  • Cryoprotectants: 20-30% glycerol or 15-20% sucrose

  • Reducing agents: 1-5 mM DTT or 2-10 mM β-mercaptoethanol (fresh)

  • Metal ions: 1-2 mM MgCl₂ to stabilize the active site

  • Additives: 0.5-1 mM EDTA to chelate heavy metals, 50-100 mM arginine to prevent aggregation

• Storage Conditions Optimization:

  Storage Method	Temperature	Concentration	Expected Stability
  Working stock	4°C	0.1-0.5 mg/ml	1-2 weeks
  Short-term	-20°C	0.5-1 mg/ml	1-3 months
  Long-term	-80°C	1-5 mg/ml	>1 year
  Lyophilized	Room temp	5-10 mg/ml	>2 years

• Stabilization Strategies:

  • Chemical modification: Cross-linking surface lysines with glutaraldehyde (0.05-0.1%)

  • Protein engineering: Introduce disulfide bonds at strategically identified positions

  • Co-factors: Include substrate analogs (acetate) at low concentrations (0.5-1 mM)

  • Carrier proteins: Add BSA (0.1-1 mg/ml) as a stabilizing carrier

  • Avoid: Repeated freeze-thaw cycles, exposure to air/oxidation, extreme pH, proteases

• Quality Control Methods:

  • Periodically test enzyme activity using the standard coupled assay

  • Monitor protein aggregation using dynamic light scattering

  • Verify secondary structure integrity using circular dichroism

  • Establish activity half-life under various storage conditions

  • Document batch-to-batch variation with standardized QC procedures

• Advanced Stabilization Technologies:

  • Immobilization on suitable matrices (Ni-NTA, CNBr-activated Sepharose)

  • Spray-dried enzyme formulations with trehalose or maltodextrin

  • Enzyme entrapment in sol-gel matrices for enhanced thermostability

  • Nanoemulsion formulations for increased shelf-life

By implementing these stability-enhancing approaches, researchers can maintain G. violaceus ackA activity at >90% for extended periods, enabling reliable experimental use and reducing the need for frequent protein preparation .

What are the critical variables that affect enzyme kinetics measurements of G. violaceus ackA?

Accurate enzyme kinetics measurements of G. violaceus acetate kinase require careful control of numerous variables that can significantly influence results. Critical factors include:

• Assay Composition Factors:

  • Buffer type and pH: Activity typically peaks at pH 7.2-7.5; deviations of 0.5 pH units can alter activity by 20-50%

  • Ionic strength: NaCl or KCl concentrations >200 mM may inhibit activity

  • Divalent cations: Mg²⁺ is essential (optimal at 2-5 mM); other divalent cations like Mn²⁺ or Ca²⁺ may compete

  • Substrate purity: Commercial acetate and ATP should be >99% pure; contaminating phosphate can inhibit the reaction

  • Coupling enzyme quality: PK and LDH must be in excess and have verified activity

• Experimental Procedure Variables:

  • Temperature control: Activity typically increases 1.5-2 fold for every 10°C increase; maintain at ±0.5°C

  • Enzyme concentration: Must be in the linear range of the assay (typically 0.5-5 μg/ml)

  • Reaction time: Initial velocity measurements should use <10% substrate conversion

  • Order of addition: Enzyme should be added last to initiate the reaction

  • Pre-incubation: Components should equilibrate to assay temperature before enzyme addition

• Instrument-Related Factors:

  • Spectrophotometer sensitivity: Should detect 0.001 absorbance unit changes reliably

  • Pathlength consistency: Use fixed pathlength cuvettes or correct for variations

  • Wavelength accuracy: Must be calibrated to measure precisely at 340 nm

  • Baseline stability: Monitor for drift over measurement period

  • Integration time: Should be optimized to reduce noise without missing kinetic events

• Data Analysis Considerations:

  • Linear range determination: Plot different enzyme concentrations vs. activity to establish linearity

  • Michaelis-Menten modeling: Use non-linear regression rather than linearization methods

  • Substrate inhibition: Test for inhibition at high substrate concentrations (>5 mM acetate or ATP)

  • Product inhibition: ADP accumulation can inhibit the reaction; keep conversion <10%

  • Statistical analysis: Calculate standard errors and confidence intervals for Km and Vmax

• Potential Interfering Factors:

  • Contaminant phosphatases may degrade ATP

  • Metal-chelating agents in buffers may reduce available Mg²⁺

  • Oxidizing agents may affect enzyme thiol groups

  • Detergents from purification may remain bound to enzyme

  • Glycerol (>5%) in enzyme storage buffer may affect kinetic measurements

Controlling these variables will ensure reproducible and accurate kinetic measurements for G. violaceus acetate kinase, allowing proper comparison with ackA enzymes from other organisms. Systematic variation of these parameters can also provide valuable insights into the unique properties of G. violaceus ackA .

What insights can comparative studies between G. violaceus ackA and other bacterial acetate kinases provide for enzyme evolution research?

Comparative studies between G. violaceus acetate kinase and other bacterial acetate kinases offer valuable insights into enzyme evolution, adaptation, and functional diversity. This research direction reveals:

• Evolutionary Trajectory Analysis:

  • G. violaceus represents one of the most ancient lineages of cyanobacteria, providing a glimpse into early enzyme evolution

  • Comparison with acetate kinases from diverse bacterial phyla can reveal ancestral features versus derived specializations

  • Identification of conserved catalytic residues across billions of years of evolution suggests fundamental mechanistic constraints

  • Divergent residues highlight adaptive responses to different ecological niches

• Structure-Function Relationship Insights:

  • Comparative structural analysis reveals how similar catalytic functions are achieved despite structural variations

  • Root Mean Square Deviation (RMSD) analysis between G. violaceus ackA and other bacterial acetate kinases (similar to the differences observed between S. aureus ackA and other bacteria with RMSD values of 1.877-2.141 Å) illuminate structural divergence patterns

  • Mapping of sequence conservation onto 3D structures identifies functionally critical regions versus plastic regions

  • Analysis of oligomerization interfaces provides insights into quaternary structure evolution

• Adaptive Specialization Evidence:

  Feature	G. violaceus ackA	Mesophilic Bacteria	Thermophilic Bacteria	Evolutionary Insight
  Temperature optima	30-40°C	25-40°C	50-80°C	Adaptation to habitat temperature
  pH optima	7.0-8.0	6.5-7.5	6.0-7.0	Adaptation to cellular pH
  Substrate specificity	Acetate-specific	Broader specificity	Narrower specificity	Specialization vs. promiscuity trade-offs
  Catalytic efficiency	Moderate	Variable	Often higher	Selection pressure differences
  Regulatory features	Unique S/T phosphorylation	Various	Minimal	Regulatory complexity evolution

• Molecular Adaptation Mechanisms:

  • Analysis of amino acid composition reveals adaptation to cellular environments (e.g., salt concentration, redox state)

  • Codon usage patterns reflect selective pressures on translation efficiency

  • Identification of convergent evolution cases where similar functional adaptations evolved independently

  • Domain architecture analysis reveals the modular nature of enzyme evolution

• Applied Evolutionary Insights:

  • Identification of catalytic plasticity hotspots amenable to enzyme engineering

  • Understanding of stability-function trade-offs guiding rational enzyme design

  • Recognition of evolutionarily conserved networks for targeted mutagenesis

  • Development of ancestral sequence reconstruction approaches to create enzymes with novel properties

These comparative studies contribute significantly to our understanding of enzyme evolution principles, providing both fundamental knowledge and practical applications for enzyme engineering. The unique position of G. violaceus in bacterial phylogeny makes its acetate kinase particularly valuable for such evolutionary studies .

How can structural information from G. violaceus ackA contribute to inhibitor design for pathogenic bacterial acetate kinases?

The structural information derived from G. violaceus acetate kinase can make significant contributions to inhibitor design targeting pathogenic bacterial acetate kinases, offering a unique perspective for antimicrobial development:

• Comparative Structural Analysis for Selective Targeting:

  • Structural comparison between G. violaceus ackA and pathogenic bacterial acetate kinases (such as those from S. aureus, M. tuberculosis) reveals both conserved and divergent regions

  • Identification of unique binding pockets or surface features in pathogenic acetate kinases that are absent in G. violaceus ackA

  • Analysis of active site architecture differences that can be exploited for selective inhibitor design

  • Determination of conformational flexibility differences that may impact inhibitor binding kinetics

• Structure-Based Virtual Screening Approaches:

  • Development of pharmacophore models based on structural differences between G. violaceus and pathogenic ackA enzymes

  • Molecular docking studies using pathogenic ackA structures with validation against G. violaceus ackA to ensure selectivity

  • Fragment-based drug design targeting unique structural elements of pathogenic acetate kinases

  • Machine learning models trained on structural features to predict selective inhibitors

• Rational Inhibitor Design Strategies:

  • Targeting the ATP binding site with modifications that exploit differences in residue composition

  • Development of transition state analogs that interact differently with G. violaceus versus pathogenic acetate kinases

  • Design of allosteric inhibitors binding to regions that affect conformational dynamics differently

  • Creation of covalent inhibitors targeting non-conserved cysteine residues in pathogenic enzymes

• Inhibitor Selectivity Analysis:

  Target Region	Conservation Status	Inhibitor Type	Selectivity Potential
  ATP binding site	Highly conserved	ATP-competitive	Low-moderate
  Acetate binding site	Moderately conserved	Substrate-competitive	Moderate
  Domain interface	Poorly conserved	Allosteric	High
  Regulatory regions	Divergent	Conformation-specific	Very high
  Surface loops	Highly divergent	Protein-protein interaction disruptors	Excellent

• Experimental Validation Approaches:

  • Parallel inhibition assays with recombinant G. violaceus ackA and pathogenic bacterial acetate kinases

  • Crystallographic studies of inhibitor binding to verify predicted binding modes

  • Isothermal titration calorimetry to compare binding thermodynamics across different acetate kinases

  • Cellular studies to confirm target engagement and antimicrobial activity

  • Resistance development monitoring to assess the evolutionary barriers to inhibitor escape

• Applied Structural Bioinformatics:

  • Multiple sequence alignment-guided identification of species-specific residues near the active site

  • Analysis of molecular dynamics trajectories to identify transiently formed pockets for inhibitor binding

  • Exploration of water-mediated interactions that differ between G. violaceus and pathogenic acetate kinases

  • Quantitative structure-activity relationship (QSAR) studies to optimize inhibitor selectivity

This structure-guided approach to inhibitor design, leveraging the unique features of G. violaceus ackA, offers promising avenues for developing targeted antimicrobial agents against pathogenic bacteria while minimizing off-target effects on beneficial microorganisms .

What emerging technologies could advance our understanding of G. violaceus ackA in vivo function?

Several cutting-edge technologies are poised to revolutionize our understanding of G. violaceus acetate kinase function in vivo, providing unprecedented insights into its cellular role:

• Advanced Imaging Technologies:

  • Cryo-electron tomography for visualizing ackA localization and interactions within the cellular environment

  • Super-resolution microscopy (PALM/STORM) combined with fluorescent protein fusions to track ackA dynamics

  • FRET-based biosensors to monitor ackA activity in real-time within living cells

  • Single-molecule tracking to analyze the mobility and clustering of ackA molecules

  • Label-free Raman microscopy to detect metabolic changes associated with ackA activity

• Multi-omics Integration Approaches:

  • Metabolic flux analysis using 13C-labeled acetate to trace ackA-dependent pathways

  • Integrative proteomics to identify the ackA interactome under various conditions

  • Phosphoproteomics to identify targets of acetyl phosphate-dependent phosphorylation

  • Transcriptomics combined with ChIP-seq to identify genes regulated by ackA activity

  • Systems biology modeling to predict emergent properties of ackA-containing networks

• Genome Engineering and Synthetic Biology Tools:

  • CRISPR interference (CRISPRi) for tunable repression of ackA expression

  • CRISPR activation (CRISPRa) for controlled upregulation of ackA

  • Optogenetic control systems for temporally precise modulation of ackA expression

  • Synthetic genetic circuits to investigate ackA regulation

  • Cell-free expression systems to study ackA activity in controlled environments

• Advanced Biochemical Approaches:

  • Protein-fragment complementation assays to map ackA interaction networks in vivo

  • Time-resolved NMR spectroscopy to capture transient enzyme states

  • Hydrogen-deuterium exchange mass spectrometry to probe conformational dynamics

  • Kinetic isotope effect studies to elucidate rate-limiting steps in the catalytic mechanism

  • Chemical cross-linking coupled with mass spectrometry to identify interaction partners

• Computational and Artificial Intelligence Methods:

  • Machine learning algorithms to predict ackA activity from multi-omics data

  • Molecular dynamics simulations with enhanced sampling to explore conformational landscapes

  • Quantum mechanics/molecular mechanics calculations to model the reaction mechanism

  • Network analysis to position ackA in the global metabolic network

  • Evolutionary coupling analysis to infer functional relationships from sequence co-evolution

• Innovative Physiological Approaches:

  • Microfluidic devices to study single-cell responses to changing acetate concentrations

  • Biosensor-equipped environmental chambers to monitor ackA activity under simulated natural conditions

  • Metabolic imaging using genetically encoded biosensors for ATP, acetate, and acetyl-CoA

  • In situ monitoring of biofilm formation dynamics in relation to ackA expression

  • Competitive fitness assays under environmentally relevant conditions

These emerging technologies, especially when used in complementary combinations, promise to transform our understanding of G. violaceus ackA from a primarily biochemical perspective to a comprehensive view of its integrated cellular functions .

What are the potential applications of engineered G. violaceus ackA variants with enhanced catalytic properties?

Engineered G. violaceus acetate kinase variants with enhanced catalytic properties offer exciting potential for diverse biotechnological applications:

• Bioenergy Applications:

  • Enhanced biofuel production through improved acetate utilization

  • Development of microbial fuel cells with increased power output via optimized ATP generation

  • Creation of artificial photosynthetic systems incorporating engineered ackA for ATP regeneration

  • Design of cellular factories for production of acetate-derived biofuels and biochemicals

  • Integration into CO2 fixation pathways to improve carbon capture efficiency

• Industrial Biocatalysis:

  • ATP regeneration systems for biocatalytic processes requiring ATP

  • Coupled enzyme systems for production of high-value acetylated compounds

  • Continuous flow bioreactors using immobilized engineered ackA

  • Thermostable variants for high-temperature industrial processes

  • pH-tolerant variants for processes under acidic or alkaline conditions

• Biosensing and Analytical Applications:

  • Development of acetate biosensors with improved sensitivity and dynamic range

  • ATP detection systems for environmental and clinical samples

  • Real-time monitoring of acetate production in fermentation processes

  • Enzyme-based analytical kits for food and beverage industry

  • Field-deployable biosensors for environmental monitoring

• Performance Enhancement Possibilities:

  Engineered Property	Potential Improvement	Key Applications
  Catalytic efficiency (kcat/Km)	5-20 fold increase	Biocatalysis, biosensing
  Thermostability	Active at 60-80°C	Industrial processes
  pH tolerance	Function at pH 4-10	Diverse reaction environments
  Substrate specificity	Accept propionate, butyrate	Expanded product range
  Oxygen tolerance	Resistance to oxidative damage	Aerobic processes
  Solvent compatibility	Function in 10-30% organic solvents	Non-aqueous reactions

• Pharmaceutical and Biomedical Applications:

  • ATP-generating systems for cell-free protein synthesis of therapeutics

  • Engineered metabolic pathways for production of acetylated pharmaceuticals

  • Acetate-removing systems for metabolic disease management

  • Enzyme replacement therapies for specific metabolic disorders

  • Diagnostic tools for acetate-producing pathogens

• Agricultural and Environmental Applications:

  • Enhanced cyanobacterial strains for biofertilizer production

  • Improved carbon fixation systems for sustainable agriculture

  • Bioremediative approaches for acetate-contaminated environments

  • Soil amendment formulations with improved nutrient cycling

  • Climate change mitigation through enhanced carbon capture

• Fundamental Research Tools:

  • Model systems to study enzyme evolution and adaptation

  • Probes for investigating metabolic regulation in complex systems

  • Tools for synthetic biology circuit design and implementation

  • Platforms for investigating metabolic flux control and optimization

  • Educational tools for demonstrating enzyme engineering principles

These applications demonstrate how protein engineering of G. violaceus ackA could translate fundamental research into diverse practical technologies with significant potential impacts across multiple sectors .