Recombinant Enterococcus faecalis Triosephosphate isomerase (tpiA)

What is triosephosphate isomerase (tpiA) and what role does it play in E. faecalis metabolism?

Triosephosphate isomerase (TIM or tpiA) is a glycolytic enzyme that catalyzes the reversible interconversion of dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate (G3P). In E. faecalis, as in other organisms, this enzyme serves as a critical component of central carbon metabolism, functioning in both glycolysis and gluconeogenesis pathways . TIM is considered a "perfectly evolved" enzyme due to its catalytic efficiency approaching the diffusion limit . In E. faecalis, which can grow in both aerobic and anaerobic conditions, tpiA likely plays an essential role in glucose metabolism across various environmental conditions, similar to other glycolytic enzymes that support this organism's metabolic flexibility.

How should researchers design expression constructs for recombinant E. faecalis tpiA?

When designing expression constructs for E. faecalis tpiA, researchers should consider:

• Expression system selection: E. coli-based expression systems are typically suitable, as demonstrated with other E. faecalis proteins .

• Tag selection: N-terminal tags like 6-His facilitate purification without interfering with enzymatic activity .

• Vector selection: pET-based vectors with T7 promoters offer high-level expression control.

• Codon optimization: Optimizing codons for E. coli expression can improve protein yields.

• Fusion partner considerations: For improved solubility, consider fusion partners similar to those used for other bacterial recombinant proteins.

The construct design should incorporate appropriate restriction sites for subsequent cloning and a protease cleavage site if tag removal is desired for functional studies.

What expression conditions optimize yield of soluble recombinant E. faecalis tpiA?

Based on protocols for other E. faecalis recombinant proteins, the following expression conditions typically yield optimal results:

These conditions have been effective for other E. faecalis enzymes and should be adaptable for tpiA expression .

What are the key structural features that characterize E. faecalis tpiA?

While the specific structure of E. faecalis tpiA has not been fully characterized, based on the highly conserved nature of triosephosphate isomerases across species, it likely exhibits:

• A classic TIM barrel fold consisting of eight α-helices surrounding eight parallel β-strands

• A homodimeric quaternary structure typical of bacterial TIMs

• Conserved catalytic residues including a glutamate that acts as a general base

• A flexible loop region (loop 6) that undergoes conformational changes during catalysis

• Structural similarity to other bacterial TIMs, particularly those from gram-positive bacteria

The enzyme likely maintains the fundamental features that make triosephosphate isomerase a benchmark of evolutionary perfection in enzyme catalysis .

How might E. faecalis tpiA function relate to virulence or biofilm formation?

While tpiA is primarily a glycolytic enzyme, it may indirectly contribute to E. faecalis virulence through several mechanisms:

• Metabolic adaptation: E. faecalis relies on glycolysis during infection of oxygen-limited environments, where tpiA activity is essential for energy generation.

• Biofilm formation support: The epa gene cluster, essential for E. faecalis biofilm formation, affects polysaccharide biosynthesis which requires metabolic precursors from glycolysis . Mutants disrupted in epa genes show decreased biofilm formation and attenuated virulence in mouse models .

• Potential moonlighting functions: Some glycolytic enzymes in other bacteria have been shown to perform secondary functions in virulence. While not directly demonstrated for E. faecalis tpiA, the presence of other moonlighting metabolic enzymes in this organism suggests similar potential.

• Metabolic coupling with virulence factors: Production of virulence factors like gelatinase (GelE) requires significant energy investment, indirectly linking glycolytic efficiency to virulence factor expression .

Research examining tpiA expression levels during biofilm formation versus planktonic growth could help elucidate these potential connections.

What approaches can be used to investigate the kinetic properties of recombinant E. faecalis tpiA?

To investigate kinetic properties of recombinant E. faecalis tpiA, researchers can employ the following methodologies:

• Coupled spectrophotometric assays: Link tpiA activity to NADH oxidation via α-glycerophosphate dehydrogenase, monitoring absorbance changes at 340 nm.

• Direct enzymatic measurements: Track the isomerization reaction using specialized techniques like circular dichroism to detect substrate-product equilibrium shifts.

• Stopped-flow kinetics: For measuring rapid reaction phases and determining microscopic rate constants.

• Temperature and pH-dependent kinetics: To determine optimum conditions and stability parameters relevant to E. faecalis environmental adaptation.

• Inhibition studies: Using known TIM inhibitors to establish structure-activity relationships specific to the E. faecalis enzyme.

A standard kinetic analysis protocol would include:

How does E. faecalis tpiA compare with homologous enzymes from other pathogens?

Comparative analysis of E. faecalis tpiA with homologs from other pathogens would likely reveal:

• Sequence conservation: The catalytic residues and TIM barrel fold are highly conserved across species, but surface residues show greater variation.

• Kinetic distinctions: E. faecalis tpiA may exhibit kinetic parameters adapted to its unique ecological niche, potentially including different substrate affinity or catalytic efficiency compared to enzymes from obligate aerobes or anaerobes.

• Structural adaptations: Subtle differences in loop regions, particularly loop 6, may reflect adaptations to different cellular environments.

• Stability characteristics: As a facultative anaerobe found in diverse environments, E. faecalis tpiA might show broader pH and temperature stability compared to more niche-specific pathogens.

• Inhibition profiles: Differential sensitivity to inhibitors could reveal species-specific binding pocket characteristics valuable for selective targeting.

Such comparative studies would provide insights into how this essential enzyme has evolved across bacterial species while maintaining its fundamental catalytic function.

What role might tpiA play in E. faecalis stress response and adaptation?

The role of tpiA in E. faecalis stress response may include:

• Metabolic flexibility: During nutrient limitation or oxidative stress, alterations in glycolytic flux through tpiA could redirect carbon to stress response pathways.

• Energy homeostasis: Maintaining ATP generation under stress conditions when other metabolic pathways are compromised.

• Cross-talk with regulatory systems: Metabolic changes sensed through glycolytic intermediates may influence expression of stress response genes, similar to connections observed between metabolism and expression of virulence factors like gelatinase .

• Adaptation to microenvironments: In biofilms, where E. faecalis experiences nutrient gradients, tpiA activity may vary across different regions, contributing to the metabolic heterogeneity that characterizes biofilm resilience .

• Contribution to antibiotic tolerance: Metabolic adaptations involving changes in glycolytic enzyme activity have been linked to antibiotic tolerance in other species, suggesting a potential similar role in E. faecalis.

Research examining tpiA expression and activity under various stress conditions would help elucidate these potential functions.

What is the optimal purification protocol for recombinant E. faecalis tpiA?

Based on successful protocols for other E. faecalis recombinant proteins, an optimal purification strategy would include:

Step 1: Cell Lysis and Clarification

• Resuspend cells in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 5% glycerol, 1 mM DTT

• Lyse cells by sonication or high-pressure homogenization

• Clarify lysate by centrifugation at 20,000 × g for 30 minutes at 4°C

Step 2: Immobilized Metal Affinity Chromatography

• Apply clarified lysate to Ni-NTA column equilibrated with lysis buffer

• Wash with 10-20 column volumes of buffer containing 20 mM imidazole

• Elute with linear gradient of 20-250 mM imidazole

Step 3: Tag Removal (Optional)

• Incubate with appropriate protease (e.g., TEV) at 4°C overnight

• Remove cleaved tag by reverse IMAC

Step 4: Size Exclusion Chromatography

• Apply concentrated protein to Superdex 75/200 column

• Elute in final buffer: 25 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol, 1 mM DTT

This protocol typically yields >95% pure protein suitable for enzymatic and structural studies .

How can researchers assay the enzymatic activity of purified E. faecalis tpiA?

For assaying E. faecalis tpiA activity, the following methods are recommended:

Method 1: Coupled Spectrophotometric Assay
Reaction components:

• 100 mM Tris-HCl, pH 7.5

• 0.2 mM NADH

• 1 mM dihydroxyacetone phosphate (DHAP)

• 1-2 U/ml α-glycerophosphate dehydrogenase

• Purified tpiA (1-10 ng)

Monitor the decrease in absorbance at 340 nm as NADH is oxidized when DHAP is converted to G3P by tpiA and then reduced by α-glycerophosphate dehydrogenase.

Method 2: Direct Assay for Forward/Reverse Reaction
For kinetic characterization, measure the equilibrium between DHAP and G3P directly:

• Using 31P NMR to track phosphorylated substrates

• Using aldehyde-specific derivatization reagents to quantify G3P formation

Method 3: O-Glycosidase Assay Adaptation
Drawing from protocols for other E. faecalis enzymes:

• Prepare reaction buffer (0.1 M MES, pH 6.0)

• Dilute enzyme to appropriate concentration (1 μg/mL)

• Add substrate and monitor product formation using appropriate detection method

• Stop reaction with NaOH and measure absorbance

Activity calculations should follow standard Michaelis-Menten analysis to determine kinetic parameters.

What strategies can improve solubility and stability of recombinant E. faecalis tpiA?

To improve solubility and stability of recombinant E. faecalis tpiA, consider:

Expression Strategies:

• Lower induction temperature (16-18°C)

• Reduce IPTG concentration (0.1-0.2 mM)

• Co-express with molecular chaperones (GroEL/GroES)

• Use auto-induction media for gradual protein expression

Buffer Optimization:

• Include stabilizing agents: 5-10% glycerol, 50-100 mM arginine

• Add reducing agents: 1-2 mM DTT or 5 mM β-mercaptoethanol

• Optimize ionic strength: typically 100-200 mM NaCl

• Test different pH values: typically 7.0-8.0 for TIM enzymes

Construct Engineering:

• Fusion partners: MBP, SUMO, or thioredoxin tags

• Surface entropy reduction: mutate surface lysine clusters

• Remove flexible termini if present

Storage Conditions:

• Flash-freeze aliquots in liquid nitrogen

• Store at -80°C with 10-15% glycerol as cryoprotectant

• Avoid repeated freeze-thaw cycles

These approaches have proven effective for other E. faecalis recombinant proteins and should be applicable to tpiA .

How can researchers investigate potential interactions between E. faecalis tpiA and other cellular components?

To investigate potential interactions between E. faecalis tpiA and other cellular components, researchers can employ:

Pull-down Assays

• Immobilize His-tagged tpiA on Ni-NTA resin

• Incubate with E. faecalis cell lysate

• Wash extensively and elute bound proteins

• Identify interacting partners by mass spectrometry

Bacterial Two-Hybrid System

• Create fusion constructs of tpiA with split reporter domains

• Screen against E. faecalis genomic library

• Validate positive interactions with complementary methods

Co-immunoprecipitation

• Generate antibodies against purified tpiA

• Perform immunoprecipitation from E. faecalis lysates

• Identify co-precipitated proteins by mass spectrometry

Crosslinking Mass Spectrometry

• Treat live E. faecalis cells with membrane-permeable crosslinkers

• Purify tpiA complexes under denaturing conditions

• Identify crosslinked peptides by specialized MS/MS analysis

Functional Interaction Screens

• Examine phenotypes of tpiA mutants in combination with other gene knockouts

• Look for synthetic lethal or suppressor interactions

• Correlate with virulence phenotypes like biofilm formation

These approaches would be particularly valuable for investigating whether tpiA interacts with virulence-associated systems like those involving enterococcal polysaccharide antigen (Epa) or gelatinase production .

How can E. faecalis tpiA be used as a model for studying enzyme evolution?

E. faecalis tpiA can serve as an excellent model for studying enzyme evolution:

• Comparative genomics: Analyze sequence conservation patterns across enterococcal species to identify conserved versus variable regions, providing insights into selective pressures.

• Ancestral sequence reconstruction: Synthesize predicted ancestral tpiA sequences to study functional changes during evolution of Enterococcus.

• Experimental evolution: Subject E. faecalis to selective pressures (temperature, pH, antibiotics) and monitor adaptive mutations in tpiA.

• Structure-function studies: Compare with triosephosphate isomerases from distantly related organisms to understand convergent evolution of catalytic mechanisms.

• Adaptation signatures: Investigate whether tpiA from clinical versus commensal E. faecalis strains shows adaptive signatures related to pathogenicity.

Triosephosphate isomerase is described as a "perfectly evolved enzyme" that very efficiently interconverts its substrates, making it an ideal subject for understanding how natural selection optimizes catalytic efficiency .

What are promising approaches for using tpiA inhibitors as potential antimicrobial agents against E. faecalis?

Approaches for developing tpiA inhibitors as antimicrobials against E. faecalis include:

• Structure-based drug design: Once the E. faecalis tpiA structure is solved, virtual screening can identify compounds that bind to catalytic or allosteric sites.

• Fragment-based approaches: Screen chemical fragment libraries for weak binders that can be elaborated into high-affinity inhibitors.

• Natural product screening: Test compounds from probiotic bacteria that naturally compete with E. faecalis for inhibitory activity against tpiA.

• Transition state analogs: Design compounds that mimic the enediol intermediate of the TIM reaction.

• Selectively targeting E. faecalis tpiA: Focus on structural differences between human and bacterial TIMs to develop selective inhibitors.

The essential nature of tpiA for glycolytic metabolism makes it a theoretically attractive target, though challenges include the high conservation of the active site and the need for selective toxicity.

How might studying E. faecalis tpiA contribute to understanding its metabolic adaptation during infection?

Studying E. faecalis tpiA can illuminate metabolic adaptation during infection through:

• Expression analysis: Quantify tpiA expression levels during different infection stages or in different host environments.

• Metabolic flux analysis: Use 13C-labeled substrates to trace carbon flow through glycolysis under infection-relevant conditions.

• In vivo studies: Create tpiA reporter strains to visualize glycolytic activity during infection in real-time.

• Host-pathogen metabolic interactions: Investigate how host metabolites affect E. faecalis tpiA activity and glycolytic flux.

• Biofilm metabolism: Compare tpiA activity in biofilm versus planktonic cells to understand metabolic shifts during biofilm formation.

This research would complement existing knowledge about E. faecalis virulence factors such as gelatinase (GelE) and enterococcal polysaccharide antigen (Epa), which are known to contribute to biofilm formation and pathogenesis .

What techniques can be used to study the role of tpiA in E. faecalis biofilm formation?

To study tpiA's role in E. faecalis biofilm formation, researchers can employ:

• Genetic approaches:

  • Create tpiA conditional knockdown strains

  • Generate site-directed mutants with altered catalytic efficiency

  • Complement mutants with wild-type tpiA to confirm phenotypes

• Biofilm assays:

  • Crystal violet staining for quantitative assessment

  • Confocal microscopy with fluorescent strains for structural analysis

  • Flow cell systems for dynamic biofilm formation studies

• Metabolic analyses:

  • Measure glycolytic intermediates in biofilm versus planktonic cells

  • Use metabolic inhibitors to assess the impact on biofilm development

  • Perform isotope tracing to track carbon flow in developing biofilms

• Integrative approaches:

  • Combine with analysis of known biofilm determinants like Epa polysaccharides

  • Investigate interactions with quorum sensing systems that regulate virulence factors

  • Study co-regulation with other metabolic pathways during biofilm formation

These approaches would build upon established methods for studying E. faecalis biofilms, which have demonstrated the importance of factors like enterococcal polysaccharide antigen in biofilm development and virulence .