Recombinant Enterococcus faecalis 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase (gpmA)

What is 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase (gpmA) and what role does it play in E. faecalis metabolism?

2,3-bisphosphoglycerate-dependent phosphoglycerate mutase (gpmA) is a key glycolytic enzyme that catalyzes the interconversion of 3-phosphoglycerate and 2-phosphoglycerate. In Enterococcus faecalis, as in many other organisms, this enzyme represents an essential step in the glycolytic pathway that ultimately leads to energy production. The dPGM variant functions through a ping-pong mechanism in which a phosphoenzyme transfers a phosphate group from an active-site histidine to 3-phosphoglycerate to form a 2,3-bisphosphoglycerate intermediate. This intermediate subsequently transfers the phosphate from the 3-position to yield the 2-phosphoglycerate product and regenerates the phosphoenzyme . As a critical metabolic enzyme, gpmA plays a vital role in E. faecalis energy production and survival.

How does E. faecalis gpmA differ from phosphoglycerate mutases in other bacterial species?

Phosphoglycerate mutases exist as non-homologous isofunctional enzymes (NISE) across bacterial species, with two main types: cofactor-dependent PGM (dPGM, like gpmA) and cofactor-independent PGM (iPGM). These enzymes have independent evolutionary origins with no similarity in primary sequence, three-dimensional structure, or catalytic mechanism . While E. faecalis primarily utilizes the dPGM form, the distribution of these enzyme types varies widely across bacteria. The PGM profile of any given bacterium is unpredictable, with some organisms such as Escherichia coli encoding both forms . The distribution is further complicated by the common occurrence of dPGM paralogs in bacterial genomes, while iPGM paralogs are relatively rare .

What is known about the genetic organization and regulation of the gpmA gene in E. faecalis?

While the search results don't provide specific information about the genetic organization of gpmA in E. faecalis, comparative analysis from other bacterial systems suggests that the expression of phosphoglycerate mutases is tightly regulated due to their essential role in central metabolism. In many bacteria, genes encoding glycolytic enzymes are often subject to carbon catabolite repression and respond to changes in nutritional status. Genome analysis indicates that the distribution of dPGM genes exhibits a patchy pattern throughout the bacterial domain, with species within the same genus, or even strains of the same species, frequently differing in their PGM repertoire . This suggests that lateral gene transfers have significantly shaped the PGM profiles across bacteria, with both intradomain and interdomain transfers apparent .

What expression systems are most effective for recombinant production of E. faecalis gpmA?

For recombinant expression of E. faecalis proteins, including gpmA, several expression systems have been developed. One particularly noteworthy system is the agmatine-inducible expression system described for E. faecalis. This system, known as the "agmatine controlled expression (ACE)" system, combines the aguR inducer gene with the P promoter (including its natural ribosome binding site) to create an effective inducible expression platform specifically designed for E. faecalis . This system offers a practical and straightforward method for heterologous protein expression in E. faecalis and has been successfully tested with green fluorescent protein (GFP) in E. faecalis V583 .

For expression in E. coli, conventional systems using T7 or similar promoters have been successfully employed for recombinant production of bacterial phosphoglycerate mutases. In a study analyzing phosphoglycerate mutases from E. coli, researchers successfully expressed and characterized both dPGM and iPGM forms, confirming they were active mutases . The specific activity of dPGM was found to greatly exceed that of iPGM, which showed highest activity in the presence of manganese .

What are the optimal purification strategies for obtaining high-yield, enzymatically active recombinant E. faecalis gpmA?

While the search results don't provide specific purification protocols for E. faecalis gpmA, general strategies for recombinant phosphoglycerate mutases typically involve affinity chromatography approaches. For instance, the pAGEnt expression vector system developed for E. faecalis was designed for the overexpression and purification of proteins fused to a 10-amino-acid His-tag at the C-terminus . This facilitates purification using nickel or cobalt affinity chromatography.

The purification protocol should consider the following methodological aspects:

• Cell lysis conditions that preserve enzyme activity (typically mild detergents or mechanical disruption)

• Buffer conditions that maintain enzyme stability (often including glycerol and reducing agents)

• Appropriate affinity tag placement that minimally affects enzymatic function

• Inclusion of the cofactor 2,3-bisphosphoglycerate during purification steps to help maintain stability

• Careful monitoring of enzymatic activity throughout the purification process

Enzymatic activity assays following purification are essential to confirm that the recombinant protein retains its functional properties, particularly its ability to catalyze the interconversion of 3-phosphoglycerate and 2-phosphoglycerate.

How can researchers optimize induction conditions when using the agmatine-inducible system for E. faecalis gpmA expression?

The agmatine-inducible expression system for E. faecalis demonstrates a close correlation between agmatine concentration and expression levels when using GFP as a reporter . To optimize induction conditions for recombinant gpmA expression, researchers should consider:

• Agmatine concentration: Under induction with 60 mM agmatine, fluorescence reached 40 arbitrary units compared to 0 in uninduced cells, indicating strong induction at this concentration .

• Growth phase: The timing of inducer addition should align with the optimal growth phase for protein expression, typically early to mid-logarithmic phase.

• Induction duration: The optimal duration for induction may vary depending on the protein and should be determined empirically.

• Temperature during induction: Lower temperatures (e.g., 25-30°C) during induction may improve protein folding and solubility.

• Media composition: Nutritional factors may affect both growth and expression efficiency.

The development of the "agmatine controlled expression (ACE)" system offers a valuable tool for E. faecalis heterologous protein expression, particularly in cases where expression in the native organism is preferred for proper folding, post-translational modifications, or functional studies .

What is the catalytic mechanism of E. faecalis gpmA and how does the requirement for 2,3-bisphosphoglycerate influence its activity?

The catalytic mechanism of dPGM enzymes, including E. faecalis gpmA, involves a ping-pong mechanism that requires 2,3-bisphosphoglycerate (2,3-BPG) as a cofactor. The mechanism proceeds as follows :

• The reaction initiates with a phosphoenzyme, in which an active-site histidine residue is phosphorylated.

• This phosphoenzyme transfers the phosphate group to 3-phosphoglycerate, forming a 2,3-bisphosphoglycerate intermediate.

• The intermediate subsequently transfers the phosphate from the 3-position to regenerate the phosphoenzyme.

• This results in the production of 2-phosphoglycerate.

The dependence on 2,3-BPG fundamentally distinguishes dPGM from iPGM variants. Without this cofactor, dPGM enzymes are catalytically inactive. The cofactor effectively serves as a "phosphate carrier" in the reaction mechanism, facilitating the transfer of the phosphate group between the active site and the substrate . This contrasts with iPGM enzymes, which catalyze a direct intramolecular phosphate transfer without requiring an external cofactor.

How do the structural features of E. faecalis gpmA compare to phosphoglycerate mutases from other organisms?

While the search results don't provide specific structural information about E. faecalis gpmA, general structural characteristics of dPGM enzymes have been extensively studied through X-ray crystallography across various bacterial species . The dPGM enzymes (like gpmA) and iPGM enzymes represent a classic example of convergent evolution, where two completely different protein structures have evolved to catalyze the same biochemical reaction.

Key structural comparisons include:

• dPGM enzymes (including gpmA) typically have a conserved protein fold that differs entirely from the fold of iPGM enzymes .

• Despite typically sharing approximately 50% sequence identity across species, dPGM enzymes maintain highly conserved active site residues and tertiary structures .

• The binding site for the 2,3-BPG cofactor is highly conserved across different dPGM variants, reflecting its essential role in catalysis.

• Both dPGM and iPGM enzymes can catalyze each other's primary reactions, albeit with decreased efficiency, highlighting how two entirely different protein structures have evolved to perform similar functions .

What factors affect the enzymatic activity and stability of recombinant E. faecalis gpmA in vitro?

Based on general principles of phosphoglycerate mutase enzymology and protein biochemistry, several factors likely influence the activity and stability of recombinant E. faecalis gpmA:

• Cofactor availability: As a dPGM, the enzyme requires 2,3-bisphosphoglycerate for activity. Enzyme preparations lacking this cofactor would show diminished or no activity .

• pH optimum: Most glycolytic enzymes have defined pH optima that reflect their intracellular environment. Deviations from this optimum can significantly affect activity and stability.

• Metal ion requirements: While dPGMs generally don't have an absolute requirement for metal ions, some divalent cations may enhance activity or stability. For comparison, iPGM showed highest activity in the presence of manganese .

• Redox environment: The presence of reducing agents may be important for maintaining critical cysteine residues in their reduced state.

• Temperature: Like most enzymes, temperature affects both the reaction rate and long-term stability of phosphoglycerate mutases.

• Substrate concentration: Kinetic parameters such as Km and Vmax would determine the enzyme's response to varying substrate concentrations.

The relative specific activity of dPGM variants is typically quite high, as demonstrated in comparative studies with E. coli, where dPGM activity greatly exceeded that of iPGM .

How did phosphoglycerate mutases evolve across bacterial species and what does this reveal about E. faecalis metabolism?

The evolution of phosphoglycerate mutases across bacterial species presents a fascinating case of non-homologous isofunctional enzymes (NISE) with independent evolutionary origins. Both cofactor-dependent (dPGM) and cofactor-independent (iPGM) forms exhibit patchy distributions throughout the bacterial domain . This distribution pattern suggests a complex evolutionary history shaped by multiple factors:

• Lateral gene transfers: Both intradomain and interdomain transfers have significantly influenced PGM profiles across bacteria .

• Gene losses and gains: The distribution is further complicated by the common occurrence of dPGM paralogs, while iPGM paralogs are rare .

• Non-orthologous gene displacement: This process can fully account for the non-uniform PGM distribution across the bacterial domain .

• Genome size correlation: Larger genomes are more likely to accommodate PGM paralogs or both NISE forms, suggesting that metabolic redundancy is a luxury afforded to organisms with greater genomic capacity .

The presence of specific PGM variants in E. faecalis likely reflects its particular metabolic needs and evolutionary history. Species within the same genus, or even strains of the same species, frequently differ in their PGM repertoire , indicating that selective pressures on glycolytic enzymes can vary even among closely related organisms.

What is known about the distribution of gpmA versus cofactor-independent phosphoglycerate mutase (iPGM) across Enterococcus species?

Based on patterns observed across other bacterial genera, we might expect:

• Variable distribution: Some Enterococcus species may encode dPGM (gpmA), others iPGM, and some might encode both forms .

• Strain-level differences: Even within a single Enterococcus species, different strains might have different PGM profiles .

• Potential paralog presence: Some Enterococcus species might harbor multiple copies of dPGM, as paralogs of this form are common in bacteria .

• Possible lateral transfers: The PGM genes in Enterococcus may have complex evolutionary histories, potentially including transfers from other bacterial genera or even other domains of life .

To establish the precise distribution across Enterococcus species, a comprehensive genomic analysis focusing specifically on this genus would be required.

How does the function of gpmA in E. faecalis compare with its role in other clinically relevant bacteria?

While the search results don't provide direct comparisons of gpmA function across different clinically relevant bacteria, we can infer some comparative aspects based on the general information provided about phosphoglycerate mutases:

• Essential metabolic role: In most bacteria, including clinically relevant species, phosphoglycerate mutases play an essential role in glycolysis, a central metabolic pathway .

• Functional redundancy: In some bacteria, such as E. coli, both dPGM and iPGM forms are present and functionally active, providing metabolic redundancy . When researchers created PGM null mutants in E. coli, they discovered that the ΔdPGM mutant grew slowly due to a delay in exiting stationary phase, but overexpression of either dPGM or iPGM overcame this defect . This suggests complementary roles in bacterial physiology.

• Potential virulence connections: Given E. faecalis's status as an opportunistic pathogen that can cause serious infections when it spreads from the gut to other parts of the body , and the essential nature of glycolytic enzymes for bacterial survival, gpmA may indirectly contribute to virulence by supporting bacterial growth and persistence during infection.

• Adaptation to environmental niches: The specific PGM profile of different bacteria may reflect adaptation to their particular environmental niches and metabolic requirements. For E. faecalis, which can transition from a commensal gut microbe to an invasive pathogen , the particular characteristics of its gpmA might reflect adaptations to these diverse environments.

What techniques are most effective for measuring the enzymatic activity of recombinant E. faecalis gpmA?

While the search results don't provide specific assay protocols for E. faecalis gpmA, standard enzymatic assays for phosphoglycerate mutase activity typically involve one of several approaches:

• Coupled enzyme assays: The production of 2-phosphoglycerate can be coupled to subsequent glycolytic enzymes (enolase, pyruvate kinase, and lactate dehydrogenase), with NADH oxidation monitored spectrophotometrically at 340 nm. This approach provides a continuous assay system that is highly sensitive.

• Direct measurement of substrate/product: High-performance liquid chromatography (HPLC) or mass spectrometry can be used to directly quantify the conversion of 3-phosphoglycerate to 2-phosphoglycerate.

• Phosphate release assays: In some experimental designs, the release or transfer of phosphate groups can be monitored using phosphate-binding molecules with spectroscopic properties.

• Isotope labeling: Using isotopically labeled substrates combined with nuclear magnetic resonance (NMR) spectroscopy can provide detailed mechanistic insights.

For recombinant E. faecalis gpmA, the assay should include the cofactor 2,3-bisphosphoglycerate, as this is essential for the activity of dPGM enzymes . Control experiments with and without the cofactor would confirm the cofactor-dependent nature of the enzyme.

How can researchers effectively design mutagenesis studies to investigate the structure-function relationships of E. faecalis gpmA?

Designing effective mutagenesis studies for E. faecalis gpmA would involve several strategic considerations:

• Target selection based on sequence conservation:

  • Identify highly conserved residues across multiple dPGM sequences, which likely play critical roles in structure or function

  • Focus on residues in the active site, particularly those involved in substrate binding or catalysis

  • Consider residues involved in binding the 2,3-bisphosphoglycerate cofactor

• Mutation types:

  • Conservative substitutions to probe subtle effects on function

  • Non-conservative substitutions to dramatically alter chemical properties

  • Alanine scanning to systematically evaluate residue contributions

• Functional analysis approaches:

  • Enzymatic activity assays comparing wild-type and mutant proteins

  • Thermal stability analysis to assess structural impacts

  • Cofactor binding studies to evaluate effects on 2,3-bisphosphoglycerate interaction

  • Crystallographic analysis of mutant proteins to directly visualize structural changes

• Expression system considerations:

  • The agmatine-inducible expression system developed for E. faecalis could be particularly valuable for expressing mutant proteins in their native cellular environment

  • Alternative expression in E. coli may provide higher yields for detailed biochemical characterization

The ping-pong mechanism of dPGM involves a phosphoenzyme intermediate, with an active-site histidine playing a crucial role . This residue would be a primary target for mutagenesis studies, along with other residues that coordinate the phosphate groups of the substrate and cofactor.

What potential exists for E. faecalis gpmA as a therapeutic target, and what approaches might be most promising?

While the search results don't directly address the therapeutic potential of E. faecalis gpmA, several factors suggest it could represent a meaningful target:

• Essential metabolic role: As a glycolytic enzyme, gpmA plays a critical role in central metabolism, making it potentially essential for bacterial survival . This is supported by observations in E. coli, where disruption of dPGM resulted in growth defects .

• Opportunistic pathogen status: E. faecalis can cause serious infections when it spreads from its normal location in the gut to other parts of the body . These infections can be life-threatening, and people with weakened immune systems, those receiving hospital treatment, those with open wounds, and several other risk groups are particularly vulnerable .

• Structural uniqueness: The distinct structural and mechanistic properties of dPGM compared to human phosphoglycerate mutases could potentially allow for selective targeting.

Promising approaches for targeting gpmA might include:

• Small molecule inhibitors: Structure-based drug design targeting the active site or cofactor binding pocket of gpmA. The required 2,3-bisphosphoglycerate cofactor presents a potential vulnerability that could be exploited .

• Allosteric modulators: Molecules that bind outside the active site but alter enzyme conformation and function.

• Gene expression interference: Approaches that reduce gpmA expression, such as antisense oligonucleotides or CRISPR interference, though these face delivery challenges.

• Combination approaches: Targeting gpmA alongside other metabolic enzymes to enhance efficacy and reduce resistance development.

The development of the agmatine-inducible expression system for E. faecalis could facilitate high-throughput screening of potential inhibitors against the native enzyme in its cellular context, potentially accelerating therapeutic discovery efforts.

What are common challenges in recombinant expression of E. faecalis gpmA and how can they be addressed?

While the search results don't specifically address challenges in E. faecalis gpmA expression, common issues in recombinant protein expression, particularly for metabolic enzymes, include:

• Protein solubility issues:

  • Challenge: Formation of inclusion bodies or aggregated protein

  • Solutions:

    • Optimize induction conditions (lower temperature, reduced inducer concentration)

    • Use solubility-enhancing fusion tags (SUMO, MBP, etc.)

    • Explore the agmatine-inducible expression system specifically developed for E. faecalis, which may provide better folding in the native cellular environment

• Cofactor incorporation:

  • Challenge: Ensuring proper incorporation of the 2,3-bisphosphoglycerate cofactor

  • Solutions:

    • Supplement expression medium or lysis buffer with the cofactor

    • Consider co-expression with enzymes that generate the cofactor

• Low expression levels:

  • Challenge: Insufficient protein yield

  • Solutions:

    • Optimize codon usage for the expression host

    • Fine-tune the concentration of agmatine when using the inducible system (60 mM agmatine showed strong induction for reporter proteins)

    • Test different promoter systems if expression is in a heterologous host

• Loss of activity during purification:

  • Challenge: Enzyme inactivation during isolation

  • Solutions:

    • Include stabilizing agents (glycerol, reducing agents) in all buffers

    • Maintain appropriate pH and ionic strength

    • Include the cofactor in purification buffers

    • Minimize temperature fluctuations and processing time

The agmatine-inducible system developed for E. faecalis has shown promise for recombinant protein expression, with GFP reporter studies demonstrating a close correlation between agmatine concentration and expression levels .

How can researchers validate that recombinantly expressed E. faecalis gpmA is properly folded and fully active?

To ensure that recombinantly expressed E. faecalis gpmA is properly folded and fully active, researchers should employ multiple validation approaches:

• Enzymatic activity assays:

  • Compare specific activity (units/mg protein) to literature values for similar enzymes

  • Determine kinetic parameters (Km, Vmax) and compare to expected values

  • Confirm the requirement for 2,3-bisphosphoglycerate, characteristic of dPGM enzymes

• Structural validation:

  • Circular dichroism (CD) spectroscopy to assess secondary structure content

  • Intrinsic tryptophan fluorescence to evaluate tertiary structure

  • Size exclusion chromatography to confirm the expected oligomeric state

  • Thermal shift assays to assess conformational stability

• Functional complementation:

  • Test whether the recombinant enzyme can rescue growth defects in PGM-deficient strains

  • In E. coli studies, overexpression of dPGM overcame growth defects in ΔdPGM mutants

• Cofactor binding:

  • Isothermal titration calorimetry (ITC) to quantify 2,3-bisphosphoglycerate binding

  • Differential scanning fluorimetry to assess thermal stabilization upon cofactor binding

• Mass spectrometry:

  • Confirm the intact mass of the purified protein

  • Analyze post-translational modifications or any chemical alterations

A combination of these approaches would provide comprehensive validation of the recombinant enzyme's integrity and functionality.

What are important considerations when designing experiments to study the biological role of gpmA in E. faecalis pathogenesis?

When designing experiments to investigate the role of gpmA in E. faecalis pathogenesis, researchers should consider several methodological aspects:

• Genetic manipulation approaches:

  • Gene deletion/knockout: Create ΔgpmA mutants, but consider potential lethality given the essential role of glycolysis

  • Conditional expression systems: The agmatine-inducible system developed for E. faecalis could allow for controlled expression

  • Point mutations: Create catalytically inactive versions to distinguish enzymatic function from potential structural roles

• Phenotypic characterization:

  • Growth kinetics under different carbon sources

  • Biofilm formation capacity

  • Resistance to environmental stresses (oxidative stress, pH, etc.)

  • Intracellular survival in host cells

  • Virulence in animal infection models

• Metabolic profiling:

  • Measure glycolytic flux using isotope-labeled glucose

  • Quantify intracellular metabolites using mass spectrometry

  • Determine changes in energy charge (ATP/ADP ratio)

• Context-specific considerations:

  • E. faecalis transitions from a commensal gut microbe to an opportunistic pathogen when it spreads to other body sites

  • Study gpmA function under conditions mimicking different infection sites (urinary tract, bloodstream, wounds)

  • Consider host factors that might interact with or modulate gpmA function

• Redundancy assessment:

  • Investigate whether E. faecalis possesses both dPGM and iPGM, as some bacteria encode both forms

  • If both are present, determine their relative contributions to metabolism and pathogenesis

• Translational potential:

  • Evaluate whether gpmA inhibition sensitizes E. faecalis to existing antibiotics

  • Assess the impact of metabolic perturbation on virulence factor expression