Recombinant Shewanella baltica Triosephosphate isomerase (tpiA)

What is the fundamental role of triosephosphate isomerase in Shewanella baltica metabolism?

Triosephosphate isomerase (tpiA) catalyzes the reversible interconversion of dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde 3-phosphate (G3P) in the glycolytic pathway. In Shewanella baltica, this enzyme plays a critical role in central carbon metabolism, particularly important for the organism's adaptation to cold environments. S. baltica, being the dominant bacterium causing seafood spoilage, relies on efficient glycolytic pathways for energy production under variable temperature conditions . The enzyme's activity is closely tied to the organism's ability to metabolize various carbon sources, which contributes to its spoilage activity in seafood products.

What expression systems are recommended for producing recombinant S. baltica tpiA?

Based on established protocols for recombinant S. baltica proteins, Escherichia coli is the preferred expression system. Similar to the approach used for other S. baltica proteins, the gene encoding tpiA should be cloned into expression vectors containing appropriate tags (typically His-tag) for purification purposes . The expression can be optimized using BL21(DE3) or Rosetta strains of E. coli, with induction typically performed using IPTG (0.5-1.0 mM) when cultures reach mid-log phase (OD₆₀₀ of 0.6-0.8). Cultivation temperature after induction should be lowered to 16-23°C to improve protein solubility, particularly important for cold-adapted enzymes like those from S. baltica.

How can the purity and identity of recombinant S. baltica tpiA be verified?

The purity and identity verification involves multiple analytical techniques:

• SDS-PAGE: Run purified protein samples on 12-15% gels to verify size and purity (>90% purity is generally required for enzymatic studies)

• Western Blotting: Use anti-His antibodies if a His-tag approach was employed

• Mass Spectrometry: For precise molecular weight determination and peptide fingerprinting

• Activity Assay: Spectrophotometric measurement of tpiA activity using coupled enzyme assays that track NADH oxidation at 340 nm

• Protein Concentration: Determine using Qubit Protein assay kits or Bradford method

Each batch should undergo quality control testing with acceptance criteria including >90% purity by SDS-PAGE, correct molecular weight verification, and specific enzymatic activity within 10% of established standards.

What are the optimal storage conditions for maintaining S. baltica tpiA stability?

For long-term storage of recombinant S. baltica tpiA, the following conditions are recommended:

When reconstituting lyophilized protein, centrifuge the vial briefly before opening to bring contents to the bottom. Reconstitute in deionized sterile water to 0.1-1.0 mg/mL concentration. Add glycerol to a final concentration of 20-50% if freezing for future use . Repeated freeze-thaw cycles should be strictly avoided as they significantly reduce enzymatic activity.

How does environmental adaptation influence the kinetic properties of S. baltica tpiA compared to mesophilic homologs?

S. baltica tpiA represents an excellent model for studying cold adaptation in enzymes. Research comparing S. baltica tpiA with mesophilic homologs reveals several distinctive adaptations:

• Lower activation energy (Ea) for catalysis at lower temperatures

• Higher catalytic efficiency (kcat/KM) at 4-15°C compared to mesophilic versions

• Increased structural flexibility, particularly around active site regions

• Reduced number of proline residues in loop regions

• Increased surface hydrophilicity

These adaptations allow S. baltica to thrive in cold marine environments and contribute to its role in seafood spoilage at refrigeration temperatures . To methodologically investigate these properties, researchers should conduct temperature-dependent kinetic studies (4-37°C range) using coupled spectrophotometric assays. Thermal stability studies using differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy are essential for correlating structural features with temperature adaptation. The data should be analyzed using Arrhenius plots to determine activation energies and transition temperatures.

What site-directed mutagenesis approaches are most effective for structure-function studies of S. baltica tpiA?

For structure-function studies of S. baltica tpiA, site-directed mutagenesis should target:

• Catalytic residues (typically Glu165 and His95, based on canonical tpiA numbering)

• Substrate binding residues

• Interface residues involved in dimerization

• Residues unique to psychrophilic tpiA variants

The QuikChange mutagenesis protocol is recommended, with some modifications for GC-rich regions that might be present in S. baltica genes. Multiple mutations should be introduced sequentially rather than simultaneously to avoid PCR complications. After mutagenesis, verify all constructs by sequencing before expression.

Analysis methodology should include:

• Enzyme kinetics comparison between wild-type and mutant proteins

• Structural analysis using X-ray crystallography or homology modeling

• Molecular dynamics simulations to understand the effect of mutations on protein flexibility

• Thermal stability measurements using DSC or thermal shift assays

The impact of mutations on cold adaptation can provide insights into the molecular basis of environmental adaptation in S. baltica, which could have implications for understanding its ecological role in marine environments and its genomic adaptation over time .

How can transcriptomic and proteomic approaches be integrated to understand the regulation of tpiA expression in S. baltica under different environmental conditions?

Integrating transcriptomic and proteomic approaches to study tpiA regulation requires a comprehensive methodology:

Experimental Design:

• Culture S. baltica under varied conditions (temperature range 4-25°C, different carbon sources, oxygen levels, and osmotic conditions)

• Extract RNA for RNA-Seq analysis and proteins for proteomics at different growth phases

• Perform quantitative RT-PCR for targeted validation of tpiA transcript levels

• Use mass spectrometry-based proteomics to quantify tpiA protein abundance

Data Integration Framework:

• Normalize transcriptomic and proteomic datasets using appropriate statistical methods

• Calculate protein-to-mRNA ratios to identify post-transcriptional regulation

• Perform gene set enrichment analysis to identify co-regulated pathways

• Construct regulatory networks using algorithms such as WGCNA (Weighted Gene Co-expression Network Analysis)

This approach would reveal how environmental conditions affect tpiA expression patterns. For example, research on S. baltica has shown that sigma factor RpoS regulates 397 differentially expressed genes related to flagellar assembly, fatty acid metabolism/degradation, and RNA degradation pathways, which are associated with cold adaptation . Similar regulatory mechanisms might affect tpiA expression during cold adaptation, potentially influencing the organism's metabolic efficiency at low temperatures.

What are the challenges in crystallizing S. baltica tpiA and how can they be overcome?

Crystallizing cold-adapted enzymes like S. baltica tpiA presents unique challenges:

Common Challenges:

• Higher structural flexibility impeding crystal formation

• Temperature-dependent conformational heterogeneity

• Solubility issues during concentration

• Limited stability during crystallization trials

Methodological Solutions:

• Temperature Screening: Perform crystallization trials at multiple temperatures (4°C, 10°C, 16°C, and 20°C), as cold-adapted proteins may adopt different conformations at different temperatures

• Additives: Include osmolytes (glycerol, trehalose) as stabilizing agents in crystallization buffers

• Ligand Co-crystallization: Attempt co-crystallization with substrate analogs or inhibitors to stabilize active site conformation

• Surface Engineering: Consider surface entropy reduction (SER) by mutating flexible surface residues to alanines

• Crystallization Techniques: Utilize microseeding and counter-diffusion methods for better crystal quality

Crystallization Screening Strategy:
Start with commercial sparse matrix screens at 4°C and 16°C, followed by optimization of promising conditions using additive screens. For diffraction data collection, crystals should be flash-cooled in liquid nitrogen using appropriate cryoprotectants like 25-30% glycerol or ethylene glycol.

How can molecular dynamics simulations contribute to understanding the cold adaptation mechanisms of S. baltica tpiA?

Molecular dynamics (MD) simulations offer powerful insights into the molecular basis of cold adaptation in S. baltica tpiA:

Simulation Setup:

• Build homology models of S. baltica tpiA if crystal structures are unavailable

• Perform comparative modeling using tpiA structures from mesophilic and thermophilic organisms

• Set up explicit solvent simulations at multiple temperatures (4°C, 15°C, 25°C, 37°C)

• Run long simulations (>100 ns) to capture relevant conformational changes

Analysis Methodology:

• Root Mean Square Fluctuation (RMSF) analysis to identify regions of increased flexibility

• Essential dynamics analysis to identify dominant motions

• Hydrogen bond and salt bridge analysis to examine differences in stabilizing interactions

• Water interaction analysis to understand solvation patterns unique to cold-adapted enzymes

• Free energy calculations to estimate stability differences at various temperatures

MD simulations can reveal how S. baltica tpiA achieves catalytic efficiency at low temperatures through specific structural adaptations. This approach complements experimental studies and can guide the design of site-directed mutagenesis experiments to verify computational findings.

What are the common expression and purification challenges for recombinant S. baltica tpiA and how can they be addressed?

Researchers frequently encounter several challenges when expressing and purifying recombinant S. baltica tpiA:

When troubleshooting purification, always perform small-scale test expressions with multiple constructs and conditions before scaling up. For storage, add 6% trehalose and maintain at -20°C/-80°C to preserve activity, with aliquoting necessary for multiple uses to avoid freeze-thaw cycles .

How can enzymatic activity assays for S. baltica tpiA be optimized for various experimental conditions?

Optimizing enzymatic activity assays for S. baltica tpiA requires careful consideration of reaction conditions:

Coupled Assay System:
The standard assay couples tpiA activity with α-glycerophosphate dehydrogenase (GDH) and measures NADH oxidation spectrophotometrically at 340 nm. For optimal results:

• Buffer Composition:

  • 100 mM Tris-HCl (pH 7.5-8.0)

  • 10 mM MgCl₂

  • 0.5 mM EDTA

  • 5 mM DTT (to maintain reduced enzyme state)

• Temperature Considerations:

  • Conduct assays at multiple temperatures (4°C, 10°C, 15°C, 25°C)

  • Pre-equilibrate all reagents to assay temperature

  • Use temperature-controlled spectrophotometer

• Substrate Concentration Range:

  • For KM determination: 0.05-5 mM substrate (8-10 concentrations)

  • For routine activity: 1 mM substrate (saturating concentration)

• Data Collection:

  • Monitor initial reaction rates (first 10% of substrate conversion)

  • Measure at least in triplicate

  • Include enzyme-free controls

• Optimization Tips:

  • Validate coupling enzyme excess (GDH) to ensure it's not rate-limiting

  • Test NADH concentration (typically 0.15-0.2 mM)

  • Optimize enzyme amount to obtain linear rates

Alternative Direct Assay:
For situations where the coupled assay is problematic, a direct assay can be developed using aldolase to cleave fructose 1,6-bisphosphate, generating DHAP that can be isomerized by tpiA. The disappearance of DHAP can be monitored using hydrazine derivatization.

What bioinformatic approaches can reveal the evolutionary adaptations of S. baltica tpiA?

Comprehensive bioinformatic analysis of S. baltica tpiA can reveal evolutionary adaptations and functional insights:

Sequence Analysis Pipeline:

• Retrieve tpiA sequences from S. baltica strains and related Shewanella species

• Include tpiA sequences from organisms across temperature ranges (psychrophilic, mesophilic, thermophilic)

• Perform multiple sequence alignment using MUSCLE or MAFFT

• Identify conserved catalytic residues and cold-adaptation signatures

• Calculate amino acid composition bias and identify substitution patterns

Structural Bioinformatics:

• Generate homology models using SWISS-MODEL or MODELLER

• Analyze electrostatic surface potentials using APBS

• Calculate accessible surface area and hydrophobicity profiles

• Perform in silico stability predictions using FoldX

• Conduct normal mode analysis to identify flexibility differences

Evolutionary Analysis:

• Construct phylogenetic trees using Maximum Likelihood or Bayesian methods

• Perform selection pressure analysis (dN/dS ratios) to identify positively selected sites

• Use ancestral sequence reconstruction to track evolutionary trajectories

• Apply coevolutionary analysis to identify functionally coupled residues

This approach can reveal how S. baltica tpiA has evolved specific adaptations related to its environmental niche. The genome sequencing of five S. baltica strains recovered from the same sample and 12 years apart from the same sampling station provides valuable resources for assessing environmental influences on genome adaptation, which may include adaptations in metabolic enzymes like tpiA .

How can differential scanning calorimetry (DSC) and circular dichroism (CD) be applied to study the thermal stability of S. baltica tpiA?

DSC and CD spectroscopy provide complementary information about the thermal stability and structural characteristics of S. baltica tpiA:

DSC Methodology:

• Sample Preparation:

  • Protein concentration: 0.5-1.0 mg/mL in phosphate buffer

  • Degassing to prevent bubble formation during heating

  • Reference cell preparation with identical buffer

• Experimental Parameters:

  • Temperature range: 0-90°C

  • Scan rate: 1°C/min for high resolution

  • Pre- and post-transition baselines: ≥10°C

  • Multiple scans to test reversibility

• Data Analysis:

  • Determine melting temperature (Tm)

  • Calculate calorimetric enthalpy (ΔHcal)

  • Assess scan reversibility to evaluate aggregation

  • Compare with mesophilic tpiA homologs

CD Spectroscopy Methodology:

• Sample Preparation:

  • Protein concentration: 0.1-0.2 mg/mL for far-UV CD

  • Buffer: 10 mM phosphate (pH 7.5) with minimal chloride

• Experimental Parameters:

  • Wavelength scan: 190-260 nm for secondary structure

  • Temperature ramps: 0.5-1°C/min from 4-90°C

  • Fixed wavelength monitoring (222 nm) during thermal denaturation

• Data Analysis:

  • Secondary structure estimation using CDNN or BeStSel

  • Thermal transition midpoint determination

  • Cooperativity of unfolding

  • Thermal stability curves comparison

These techniques should reveal the thermodynamic parameters associated with S. baltica tpiA stability and provide insights into how this cold-adapted enzyme maintains activity at lower temperatures while sacrificing stability at higher temperatures, a hallmark of psychrophilic adaptations.

How can CRISPR-Cas9 genome editing be applied to study tpiA function in S. baltica?

CRISPR-Cas9 technology offers powerful approaches for studying tpiA function directly in S. baltica:

Methodological Framework:

• Design of sgRNA targeting tpiA:

  • Identify PAM sites within the tpiA gene

  • Design guide RNAs with minimal off-target effects

  • Validate guide efficiency in silico using tools like CHOPCHOP

• Delivery Methods for S. baltica:

  • Conjugation-based transfer of CRISPR plasmids

  • Electroporation optimization for S. baltica (buffer composition, field strength)

  • Temperature-sensitive plasmids for transient expression

• Genome Editing Strategies:

  • Gene knockout: introduce frameshift mutations

  • Point mutations: use homology-directed repair with repair templates

  • Promoter modification: alter expression levels rather than sequence

  • Protein tagging: introduce fluorescent or affinity tags

• Phenotypic Analysis:

  • Growth rate determination under various conditions

  • Metabolic profiling using LC-MS

  • Cold adaptation assays (4-15°C)

  • Comparative transcriptomics of wild-type vs. mutants

This approach would allow direct investigation of tpiA's role in S. baltica metabolism and cold adaptation. The findings could be related to broader genomic adaptation patterns observed across S. baltica strains collected from the same environments over time, as documented in previous genomic studies .

What is the potential role of S. baltica tpiA in developing cold-active enzyme applications?

S. baltica tpiA represents a promising candidate for cold-active enzyme applications due to its adaptation to low-temperature environments:

Potential Applications:

• Biocatalysis in Cold Conditions:

  • Low-temperature food processing

  • Cold-wash detergent formulations

  • Bioremediation in cold environments

• Structural Template for Enzyme Engineering:

  • Rational design of cold-active variants of industrial enzymes

  • Identification of flexibility-enhancing mutations

• Metabolic Engineering Platform:

  • Enhancement of glycolytic flux at low temperatures

  • Development of cold-adapted microbial cell factories

Research Methodology for Application Development:

• Enzyme Engineering Approach:

  • Directed evolution under selective pressure

  • Structure-guided design of stability-flexibility balance

  • High-throughput activity screening at various temperatures

• Formulation Development:

  • Stability enhancement through excipient screening

  • Immobilization strategies for improved reusability

  • Compatibility testing with industrial processes

• Performance Metrics:

  • Activity measurements at 0-25°C range

  • Long-term stability assessment

  • Comparison with commercial alternatives

The unique cold-adaptive features of S. baltica enzymes that enable its growth and metabolic activity at low temperatures, including its role in seafood spoilage , make tpiA particularly interesting for biotechnological applications requiring enzymatic activity in cold conditions.

How does the interactome of S. baltica tpiA contribute to its cellular function and cold adaptation?

Understanding the protein interaction network (interactome) of S. baltica tpiA provides insights into its cellular context and cold adaptation mechanisms:

Methodological Approaches:

• Affinity Purification-Mass Spectrometry (AP-MS):

  • Tag tpiA with affinity tags (His, FLAG, etc.)

  • Express in native S. baltica

  • Purify under gentle conditions to maintain interactions

  • Identify interacting partners by mass spectrometry

• Yeast Two-Hybrid Screening:

  • Use tpiA as bait against S. baltica genomic DNA library

  • Screen for interactions at low temperature (16-20°C)

  • Validate interactions with co-immunoprecipitation

• Proximity Labeling:

  • Fuse tpiA with BioID or APEX2

  • Express in S. baltica and activate labeling

  • Identify proximal proteins by streptavidin pull-down and MS

• Co-expression Network Analysis:

  • Analyze transcriptomic data across multiple conditions

  • Identify genes co-regulated with tpiA

  • Construct functional association networks

Expected Interactome Components:

• Other glycolytic enzymes forming metabolons

• Chaperones involved in cold adaptation

• Regulatory proteins responding to temperature shifts

• Membrane proteins for metabolite transport

How does S. baltica tpiA compare structurally and functionally to tpiA from other psychrophilic, mesophilic, and thermophilic organisms?

Comparative analysis of tpiA across temperature-adapted organisms provides fundamental insights into enzymatic cold adaptation:

Comparative Analysis Framework:

Methodological Approach for Comparison:

• Recombinantly express and purify tpiA from representative organisms

• Conduct temperature-dependent kinetic analysis (4-80°C)

• Perform structural characterization using X-ray crystallography or homology modeling

• Analyze local and global flexibility using hydrogen-deuterium exchange mass spectrometry

• Conduct comparative molecular dynamics simulations

These comparisons would reveal the molecular basis for cold adaptation in S. baltica tpiA, potentially including increased active site flexibility, reduced structural stabilizing interactions, and optimized surface properties for function at low temperatures.

What insights can comparative genomics provide about the evolution of tpiA in S. baltica strains from different environmental conditions?

Comparative genomics offers valuable insights into how tpiA has evolved in S. baltica strains adapted to different environmental niches:

Research Methodology:

• Sequence Collection and Analysis:

  • Retrieve tpiA sequences from all sequenced S. baltica strains

  • Include strains from different environmental conditions and time points

  • Perform multiple sequence alignment and identify polymorphic sites

• Phylogenetic Analysis:

  • Construct phylogenetic trees using Maximum Likelihood

  • Map environmental metadata onto phylogenetic trees

  • Identify environment-specific clustering

• Selection Analysis:

  • Calculate dN/dS ratios across the gene

  • Identify sites under positive, negative, or relaxed selection

  • Correlate selection patterns with environmental variables

• Genomic Context Analysis:

  • Examine conservation of gene neighborhood

  • Identify potential horizontal gene transfer events

  • Analyze promoter regions for regulatory differences