Recombinant Shewanella baltica Glucose-6-phosphate isomerase (pgi), partial

What is Shewanella baltica Glucose-6-phosphate isomerase and what is its function?

Glucose-6-phosphate isomerase (GPI), also known as phosphoglucose isomerase (PGI) or phosphohexose isomerase (PHI), is an enzyme with EC classification 5.3.1.9 that catalyzes the reversible conversion of glucose-6-phosphate to fructose-6-phosphate, a critical step in both glycolysis and gluconeogenesis pathways. In Shewanella baltica, this enzyme facilitates carbohydrate metabolism under psychrotrophic conditions. The enzyme has been observed to possess dual functionality, with both isomerase activity and lysyl aminopeptidase (PGI-LysAP) activity . This bifunctional nature may contribute to S. baltica's metabolic flexibility in cold marine environments.

What are the optimal storage conditions for Recombinant Shewanella baltica GPI?

For optimal stability and activity preservation, recombinant S. baltica GPI should be stored at -20°C for regular storage periods, while extended storage requires temperatures of -20°C or -80°C . It is strongly recommended to avoid repeated freeze-thaw cycles as they can significantly compromise protein integrity and enzymatic activity. For working solutions, aliquots may be maintained at 4°C for up to one week . When preparing for long-term storage, addition of glycerol as a cryoprotectant (typically to a final concentration of 50%) is advisable prior to freezing .

How should Recombinant Shewanella baltica GPI be reconstituted for experimental use?

The recommended reconstitution protocol involves briefly centrifuging the protein vial before opening to ensure content collects at the bottom. The lyophilized protein should be reconstituted in deionized sterile water to achieve a concentration between 0.1-1.0 mg/mL . For optimal stability during long-term storage, glycerol should be added to a final concentration of 5-50% (with 50% being standard practice) before aliquoting and storing at -20°C or -80°C . This approach minimizes activity loss during storage while maintaining the protein in a form readily available for experimental applications.

What growth characteristics of Shewanella baltica impact research with its recombinant proteins?

Shewanella baltica exhibits distinct growth characteristics that researchers should consider when working with its recombinant proteins:

• Temperature sensitivity: S. baltica has an optimal growth temperature around 25°C and does not grow well at 37°C, requiring lower temperature incubations for cultivation .

• Psychrotrophic adaptation: The organism can grow at temperatures as low as 0°C, reflecting evolutionary adaptations to cold marine environments .

• Respiratory versatility: Being a facultative anaerobe, S. baltica can grow in both aerobic and anaerobic conditions, which may influence protein expression profiles and enzyme characteristics.

• Marine adaptation: As a marine bacterium, S. baltica is adapted to specific salt concentrations, which may affect the stability and activity of its native enzymes including GPI.

These characteristics suggest that recombinant S. baltica proteins, including GPI, may exhibit optimal activity under conditions that differ from standard laboratory protocols designed for mesophilic organisms.

How does the enzymatic activity of Recombinant Shewanella baltica GPI compare to GPI from mesophilic organisms?

The enzymatic characteristics of S. baltica GPI likely reflect adaptations to cold environments:

• Temperature-activity profile: S. baltica GPI would be expected to maintain higher relative activity at lower temperatures (10-25°C) compared to mesophilic homologs, which typically exhibit activity optima around 37°C.

• Catalytic efficiency (kcat/Km): At lower temperatures, S. baltica GPI may demonstrate higher catalytic efficiency than mesophilic counterparts, compensating for the reduced molecular kinetic energy available.

• Conformational flexibility: Cold-adapted enzymes often exhibit increased flexibility around active sites, achieved through fewer rigid structural elements and intramolecular hydrogen bonds.

• Thermostability trade-off: S. baltica GPI likely demonstrates lower thermal stability than mesophilic versions, representing an evolutionary trade-off that favors activity at lower temperatures over structural rigidity.

• Activation energy: The activation energy requirement for S. baltica GPI-catalyzed reactions may be lower, facilitating catalysis under conditions of reduced thermal energy.

Experimental approaches to investigate these differences should include comparative enzyme kinetics across temperature ranges (0-50°C), thermal inactivation studies, and structural dynamics analyses using techniques such as hydrogen-deuterium exchange mass spectrometry.

What role might GPI play in the quorum sensing systems of Shewanella baltica?

Recent research has established that Shewanella baltica employs sophisticated quorum sensing (QS) systems that regulate spoilage potential and biofilm formation . While direct evidence linking GPI activity to QS is not explicitly established in current literature, several potential connections merit investigation:

• Metabolic integration: GPI's central role in carbohydrate metabolism may influence the availability of metabolic precursors required for QS signaling molecule synthesis, particularly diketopiperazines like cyclo-(L-Pro-L-Phe) (PP) .

• Regulatory network overlap: Transcriptional regulation of GPI might be coordinated with QS-regulated genes, especially under stress conditions where metabolic remodeling occurs simultaneously with community behavior changes.

• Biofilm contribution: GPI activity could influence exopolysaccharide production or composition, which is a critical component of biofilm matrices. The spoilage-related genes regulated by the LuxR-type QS system in S. baltica OS155 include torS, speF, and pomA , which could have indirect metabolic connections to GPI activity.

• Environmental adaptation: Both QS systems and GPI activity are responsive to environmental conditions, suggesting possible co-regulation during cold adaptation or nutrient limitation scenarios.

Research approaches to explore these connections should include transcriptomic and proteomic comparisons between wild-type and QS-deficient mutants, metabolic flux analysis under QS-active and QS-inhibited conditions, and investigation of GPI activity in the presence of isolated QS signaling molecules.

How can researchers characterize the dual PGI-LysAP functionality of Shewanella baltica GPI?

The dual functionality of S. baltica GPI, possessing both glucose-6-phosphate isomerase and lysyl aminopeptidase activities , presents unique research opportunities:

• Activity separation strategies:

  • pH optimization experiments to identify differential pH optima for each activity

  • Selective inhibition studies using activity-specific inhibitors

  • Substrate competition assays to determine interaction between binding sites

• Structural investigation approaches:

  • X-ray crystallography with various substrates and substrate analogs

  • Site-directed mutagenesis targeting predicted catalytic residues for each activity

  • Protein truncation experiments to identify minimal domains required for each function

• Evolutionary analysis:

  • Comparative genomics across Shewanella species to trace the acquisition of dual functionality

  • Phylogenetic analysis to determine if this represents convergent or divergent evolution

  • Structural comparison with single-function homologs from related organisms

• Methodological considerations:

  • Development of high-throughput assays capable of simultaneously monitoring both activities

  • Kinetic analysis under various temperature conditions relevant to S. baltica's environmental niche

  • Protein engineering approaches to selectively enhance or suppress each activity

This dual functionality may represent an evolutionary adaptation that provides metabolic efficiency in resource-limited cold environments.

What structural features might contribute to the cold adaptation of Shewanella baltica GPI?

Cold-adapted enzymes typically display structural modifications that enhance flexibility and catalytic efficiency at low temperatures. For S. baltica GPI, these features likely include:

Experimental approaches to investigate these features should combine high-resolution structural studies (X-ray crystallography, cryo-EM) with dynamics assessments (hydrogen-deuterium exchange, molecular dynamics simulations) and comparative analyses with mesophilic and thermophilic GPI homologs.

What are the optimal assay conditions for measuring Shewanella baltica GPI enzymatic activity?

Given the psychrotrophic nature of Shewanella baltica, optimal assay conditions for its GPI should be carefully established:

• Temperature considerations:

  • Primary activity measurements should be conducted at 25°C, corresponding to S. baltica's optimal growth temperature

  • Comparative assays at 4°C, 15°C, and 37°C to characterize temperature dependence

  • Temperature equilibration of all reagents before assay initiation

• Buffer system optimization:

  • pH range: 6.5-8.0 (MOPS or phosphate buffer systems)

  • Salt concentration: 100-200 mM NaCl to mimic marine conditions

  • Potential cofactors: Test inclusion of 0.5-1 mM MgCl₂ or other divalent cations

  • Buffer capacity adequate to prevent pH shifts during reaction

• Substrate parameters:

  • Concentration range: 0.1-10 mM glucose-6-phosphate for forward reaction

  • For reverse direction: 0.1-10 mM fructose-6-phosphate

  • Substrate purity verification to eliminate interferents

• Detection methods:

  • Continuous spectrophotometric assay coupling with glucose-6-phosphate dehydrogenase and NADP⁺

  • Endpoint assays with colorimetric detection of phosphorylated sugars

  • HPLC-based methods for direct product quantification

• Quality control measures:

  • Inclusion of appropriate enzyme-free and substrate-free controls

  • Commercial GPI standards for comparative analysis

  • Linearity verification across the measurement range

Activity should be expressed in μmol of substrate converted per minute per mg of enzyme under defined conditions, with careful documentation of all parameters to ensure reproducibility.

How can isotope labeling approaches be used to study Shewanella baltica GPI in metabolic networks?

Isotopic labeling provides powerful tools for investigating GPI's role within cellular metabolism:

• Substrate labeling strategies:

  • [1-¹³C]glucose or [6-¹³C]glucose to trace specific carbon routing

  • [U-¹³C]glucose for comprehensive pathway mapping

  • [²H] or [¹⁸O] labeled substrates to follow hydrogen or oxygen transfer

• Experimental approaches:

  • Time-course sampling to capture metabolic dynamics

  • Temperature shift experiments to assess cold adaptation effects

  • Comparison between wild-type and GPI-modified strains

• Analytical methodologies:

  • LC-MS/MS for detection and quantification of labeled metabolites

  • NMR spectroscopy for positional isotope analysis

  • GC-MS for volatile derivatives after sample preparation

• Data analysis techniques:

  • Isotopomer distribution analysis using specialized software

  • Metabolic flux analysis (MFA) for quantitative pathway mapping

  • Integration with genome-scale metabolic models of S. baltica

• Experimental considerations:

  • Rapid quenching of metabolism to capture accurate snapshots

  • Efficient extraction protocols for phosphorylated intermediates

  • Appropriate controls for natural isotope abundance correction

This approach can reveal how GPI activity changes under different environmental conditions and how carbon flux through glycolysis is regulated in S. baltica, particularly during cold adaptation responses.

What approaches can be used to study the interaction between Shewanella baltica GPI and potential protein partners?

Investigating protein-protein interactions involving S. baltica GPI requires approaches adapted to its properties:

• In vitro interaction methods:

  • Pull-down assays using epitope-tagged recombinant GPI

  • Surface plasmon resonance at temperatures relevant to S. baltica (10-25°C)

  • Isothermal titration calorimetry for quantitative binding parameters

  • Chemical cross-linking coupled with mass spectrometry for interaction site mapping

• Structural biology approaches:

  • Co-crystallization attempts with predicted interaction partners

  • Cryo-electron microscopy of protein complexes

  • NMR titration experiments to map interaction interfaces

• In vivo techniques:

  • Bacterial two-hybrid systems adapted for lower temperature operation

  • Co-immunoprecipitation from S. baltica lysates under native conditions

  • Fluorescence microscopy to detect co-localization patterns

  • In vivo crosslinking to capture transient interactions

• Bioinformatic predictions:

  • Analysis of gene proximity and co-expression patterns

  • Protein-protein interaction prediction algorithms

  • Structural docking simulations with potential partners

• Functional validation:

  • Activity assays in the presence of identified interaction partners

  • Mutational analysis of predicted interaction interfaces

  • Competition assays with peptides derived from interaction regions

When designing these experiments, special attention should be paid to maintaining conditions appropriate for S. baltica proteins, including reduced temperature and suitable buffer composition reflecting the marine environment.

How can researchers effectively generate and validate mutations in Shewanella baltica GPI for structure-function studies?

Systematic mutation strategies for S. baltica GPI structure-function analysis should follow these methodological guidelines:

• Rational design approaches:

  • Multiple sequence alignment with GPI from psychrophilic, mesophilic, and thermophilic organisms

  • Homology modeling to identify structurally important regions

  • Computational prediction of residues involved in cold adaptation

  • Focus on active site residues, interdomain interfaces, and surface-exposed flexible regions

• Expression system considerations:

  • Cold-adapted expression hosts (Arctic Express E. coli) for proper folding

  • Temperature-inducible promoter systems

  • Codon optimization for expression host while maintaining amino acid sequence

  • Inclusion of appropriate purification tags that minimally impact structure

• Validation approaches:

  • Thermal stability comparison (DSC or DSF) against wild-type enzyme

  • Temperature-dependent activity profiles (5-40°C)

  • Structural integrity verification via circular dichroism

  • Substrate affinity and specificity determination

  • pH-activity profiles to detect altered ionization behavior

• Advanced biophysical characterization:

  • Hydrogen-deuterium exchange mass spectrometry to assess flexibility changes

  • Molecular dynamics simulations at various temperatures

  • NMR relaxation measurements for dynamics assessment

  • X-ray crystallography of key mutants when possible

• Functional complementation tests:

  • In vivo testing in GPI-deficient bacterial strains

  • Growth rate analysis at different temperatures

  • Metabolomic profiling to detect pathway alterations

A comprehensive approach would involve multiple mutation strategies, including both site-directed changes targeting specific hypotheses and broader scanning approaches to identify unexpected determinants of cold adaptation.

How can researchers reconcile contradictory findings in studies of Shewanella baltica GPI activity?

When confronted with conflicting experimental results, a systematic analytical framework should be employed:

• Methodological variance analysis:

  • Examination of buffer composition differences (pH, ionic strength, additives)

  • Temperature control precision across studies (even 2-3°C can significantly affect cold-adapted enzymes)

  • Protein preparation methods (expression system, purification protocol, storage conditions)

  • Assay methodologies (detection systems, reaction time, substrate purity)

• Statistical evaluation approaches:

  • Reanalysis of raw data when available

  • Meta-analysis of multiple studies with appropriate weighting

  • Sensitivity analysis to identify parameters with greatest impact on outcomes

  • Consideration of statistical power and biological variability

• Strain-specific considerations:

  • Genetic verification of S. baltica strains used across studies

  • Potential strain-specific differences in post-translational modifications

  • Growth conditions prior to protein isolation

• Reconciliation strategies:

  • Development of standardized assay protocols specifically for S. baltica GPI

  • Direct side-by-side comparison under identical conditions

  • Collaborative cross-laboratory validation studies

  • Identification of environmental variables that might explain apparent contradictions

• Biological context integration:

  • Consideration of enzyme behavior in cellular context versus isolated systems

  • Evaluation of potential allosteric regulators present in some experimental systems but not others

  • Assessment of protein microheterogeneity across preparations

This systematic approach can transform apparently contradictory results into valuable insights about condition-dependent behavior of S. baltica GPI.

What statistical approaches are most appropriate for analyzing temperature-dependent kinetic data for Shewanella baltica GPI?

Temperature-dependent enzyme kinetics for psychrophilic enzymes require specialized statistical treatment:

• Model selection considerations:

  • Modified Arrhenius equations accounting for temperature-dependent changes in protein dynamics

  • Equilibrium model incorporating both catalytic effects and temperature-dependent inactivation

  • Non-linear models that account for cold and heat denaturation phenomena

• Parameter estimation methods:

  • Weighted non-linear regression accounting for heteroscedasticity across temperature range

  • Global fitting approaches for simultaneous analysis of multiple datasets

  • Bootstrap resampling for robust confidence intervals

  • Bayesian parameter estimation with suitable priors

• Comparative analysis techniques:

  • Analysis of covariance (ANCOVA) for comparing temperature dependence across variants

  • Multiple comparison corrections when testing across temperature points

  • Principal component analysis for multiparametric data

• Data visualization approaches:

  • Three-dimensional surface plots (activity-temperature-substrate concentration)

  • Activation energy profile plots across temperature ranges

  • Comparative radar plots for multiple parameters across enzyme variants

  • Residual analysis plots to identify systematic deviations from models

Specialized software tools including R packages for enzyme kinetics (drc, nlstools) or Python implementations with thermodynamic modeling capabilities are recommended for appropriate analysis.

How might recombinant Shewanella baltica GPI contribute to developing cold-active enzymes for biotechnological applications?

The study of S. baltica GPI offers several promising avenues for biotechnological development:

• Structural insights for cold-adaptation engineering:

  • Identification of specific residues and structural elements conveying cold activity

  • Understanding of flexibility-stability relationships applicable to other enzyme classes

  • Elucidation of design principles for reduced activation energy barriers

• Potential biotechnological applications:

  • Low-temperature biocatalysis for pharmaceutical and fine chemical synthesis

  • Food processing enzymes with activity during refrigeration

  • Cold-active diagnostic reagents with extended shelf-life

  • Environmental bioremediation technologies for cold climates

• Enzyme engineering strategies informed by S. baltica GPI:

  • Rational design based on comparative structural analysis

  • Directed evolution with low-temperature selection pressure

  • Domain swapping between psychrophilic and mesophilic homologs

  • Computational design incorporating molecular dynamics simulations

• Industrial process development considerations:

  • Energy savings through reduced heating requirements

  • Selective reactions at low temperatures to minimize side reactions

  • Enhanced sustainability through reduced thermal energy input

  • Novel reaction conditions enabling previously challenging transformations

Research in this area should focus on understanding the molecular basis of cold adaptation in S. baltica GPI, then applying these principles to other industrially relevant enzyme systems through protein engineering approaches.

What integrated approaches could advance our understanding of Shewanella baltica GPI in the context of marine adaptation?

Comprehensive investigation of S. baltica GPI requires multidisciplinary integration:

• Evolutionary genomics approaches:

  • Comparative analysis of GPI sequences across Shewanella species from different thermal environments

  • Reconstruction of ancestral sequences to identify adaptive mutations

  • Population genomics across marine temperature gradients

  • Assessment of horizontal gene transfer contribution to GPI diversity

• Structural biology integration:

  • High-resolution structures determined at physiologically relevant temperatures

  • Dynamics studies capturing the conformational ensemble across temperature ranges

  • Investigation of potential cold-specific conformational states

  • Structural basis for dual enzymatic activity (isomerase and aminopeptidase)

• Systems biology perspectives:

  • Integration of GPI function within genome-scale metabolic models of S. baltica

  • Multi-omics approaches (transcriptomics, proteomics, metabolomics) during temperature shifts

  • Regulatory network mapping focusing on cold-responsive elements

  • Flux balance analysis under varying environmental conditions

• Ecological contextualization:

  • Correlation of GPI activity with marine temperature gradients

  • Investigation of seasonal adaptation patterns

  • Interspecies comparison across marine psychrophiles

  • Assessment of GPI contribution to competitive fitness in cold environments

This integrated approach would provide a comprehensive understanding of how GPI function contributes to S. baltica's ecological success and adaptation to marine environments, while also advancing our fundamental knowledge of enzyme cold adaptation mechanisms.