Recombinant Enterococcus faecalis Fructose-1,6-bisphosphatase class 3 (fbp), partial

What is Fructose-1,6-bisphosphatase and what role does it play in Enterococcus faecalis metabolism?

Fructose-1,6-bisphosphatase (FBPase) is a key enzyme that governs a critical step in gluconeogenesis, catalyzing the hydrolysis of fructose 1,6-bisphosphate to form fructose 6-phosphate and inorganic phosphate. In Enterococcus faecalis, this enzyme plays a crucial role in carbohydrate metabolism, particularly when the bacterium needs to synthesize glucose from non-carbohydrate precursors. Unlike mammalian FBPases, which are subject to well-characterized metabolic regulation, the regulatory mechanisms of bacterial FBPases, including those in E. faecalis, are not as thoroughly understood . This enzyme represents an important component of the metabolic flexibility that allows E. faecalis to adapt to various nutritional environments, including nutrient-limited conditions encountered during infection and host colonization.

How does E. faecalis FBPase class 3 differ structurally from other classes of FBPases?

While the search results don't provide specific structural information about E. faecalis FBPase class 3, we can infer from related bacterial FBPases that class 3 FBPases likely exhibit significant structural differences from the better-characterized class 1 enzymes. Based on studies of prokaryotic FBPases like those found in E. coli, E. faecalis FBPase class 3 likely forms a homotetramer, but with unique quaternary structural arrangements that differ from both the canonical R- and T-states observed in mammalian FBPases . The structural differences likely extend to the active site architecture and regulatory binding pockets, which would explain the distinct regulatory mechanisms observed in bacterial versus mammalian FBPases. Methodologically, these structural differences are typically characterized through X-ray crystallography, cryo-electron microscopy, and comparative sequence analysis with other bacterial FBPases.

What expression systems are most effective for producing recombinant E. faecalis FBPase class 3?

For efficient production of recombinant E. faecalis FBPase class 3, researchers typically employ prokaryotic expression systems, predominantly E. coli strains optimized for protein expression such as BL21(DE3) or Rosetta. The methodology involves cloning the fbp gene into expression vectors containing inducible promoters (like T7 or tac) and appropriate affinity tags (His6, GST, or MBP) to facilitate purification. Temperature optimization is crucial, with expression often performed at reduced temperatures (16-25°C) to enhance proper folding. For purification, a combination of affinity chromatography, ion exchange, and size exclusion chromatography typically yields high-purity enzyme suitable for enzymatic and structural studies. When working with recombinant E. faecalis proteins, researchers must carefully optimize induction conditions and consider codon optimization due to potential codon usage bias between E. faecalis and the expression host.

How does carbohydrate metabolism in E. faecalis affect its survival within macrophages?

Recent transposon insertion sequencing studies have revealed that carbohydrate metabolism, particularly fructose and mannose metabolism, plays a significant role in E. faecalis survival within macrophages. Interestingly, attenuation of carbohydrate metabolism appears to promote E. faecalis survival in macrophages, suggesting a complex relationship between metabolism and immune evasion . E. faecalis modulates its carbohydrate metabolism to avoid activating the immune response of macrophages, which contributes to its long-term survival within these immune cells. This metabolic adaptation represents a sophisticated survival mechanism whereby E. faecalis essentially "flies under the radar" of the host immune system by reducing metabolic activities that might trigger stronger immune responses.

What are the known allosteric regulators of E. faecalis FBPase and how do they compare to regulators in other species?

While specific information about E. faecalis FBPase regulators isn't detailed in the search results, insights from prokaryotic FBPases suggest that phosphoenolpyruvate (PEP) likely serves as an allosteric activator for E. faecalis FBPase, similar to its role in E. coli FBPase where it increases activity by at least 300% . This stands in stark contrast to mammalian FBPases, which are primarily regulated by the inhibitory effects of AMP and fructose 2,6-bisphosphate. In E. coli, the binding of anionic ligands to allosteric activator sites stabilizes a tetrameric structure and a polypeptide fold that prevents AMP binding . Additionally, sulfate ions have been identified as activators of bacterial FBPases, binding to conserved residues that are present in many heterotrophic bacteria but absent in organisms that use fructose 2,6-bisphosphate as a regulator.

How does the phosphotransferase system (PTS) interact with FBPase function in E. faecalis metabolism?

The phosphotransferase system (PTS) in E. faecalis plays a critical role in carbohydrate uptake and metabolism, particularly for fructose and mannose, which directly connects to FBPase function. In E. faecalis, multiple PTS systems have been identified, with some specifically dedicated to fructose and mannose transport . The PTS system transfers phosphate from phosphoenolpyruvate (PEP) to incoming sugars, creating fructose-6-phosphate or mannose-6-phosphate, which enter the metabolic pathway where FBPase operates. Experimental evidence has shown that deletion of certain PTS components affects growth on specific sugars, highlighting the interconnection between transport and metabolism . The relationship between PTS and FBPase represents a coordinated system where sugar uptake is directly linked to its subsequent metabolism, allowing fine-tuned control of carbon flux through gluconeogenesis versus glycolysis.

What are the optimal conditions for measuring E. faecalis FBPase activity in vitro?

When measuring E. faecalis FBPase activity in vitro, researchers should optimize several key parameters. The standard assay typically couples FBPase activity to secondary enzymes (phosphoglucoisomerase and glucose-6-phosphate dehydrogenase) and monitors NADPH production at 340 nm. Optimal conditions generally include: buffer system (MOPS or Tris-HCl at pH 7.0-7.5), temperature (30-37°C), divalent cations (5-10 mM Mg²⁺ or Mn²⁺), substrate concentration (0.05-2 mM fructose 1,6-bisphosphate), and potential activators like phosphoenolpyruvate (1-5 mM) . When investigating allosteric regulation, researchers should include appropriate concentrations of hypothesized regulators such as PEP, sulfate, or AMP. For accurate kinetic analysis, initial velocity conditions must be maintained, typically by limiting reaction time and enzyme concentration. Data analysis should include Michaelis-Menten kinetics, along with appropriate models for allosteric regulation if investigating regulatory mechanisms.

What methods are most effective for studying the interaction between E. faecalis FBPase and host immune cells?

To study interactions between E. faecalis FBPase and host immune cells, researchers employ several complementary approaches. For investigating the role of FBPase in bacterial survival within macrophages, the creation of fbp gene deletion mutants using CRISPR-Cas9 or homologous recombination is essential, followed by macrophage infection assays comparing wild-type and mutant strains . Intracellular survival can be quantified through gentamicin protection assays, confocal microscopy with fluorescently-labeled bacteria, and flow cytometry. Transposon insertion sequencing (TIS) has proven valuable for identifying genes, including fbp, that affect bacterial fitness during macrophage infection . Host response can be assessed through transcriptomics, measurement of inflammatory cytokines, and analysis of macrophage metabolic shifts. Advanced techniques include isotope labeling to track carbon flux, fluorescence resonance energy transfer (FRET) to detect protein-protein interactions, and in vivo infection models using transgenic mice with labeled immune cell populations.

How can researchers effectively design inhibitors targeting E. faecalis FBPase class 3?

Designing effective inhibitors for E. faecalis FBPase class 3 requires a multidisciplinary approach integrating structural biology, computational methods, and biochemical validation. Researchers should begin with obtaining high-resolution crystal structures of E. faecalis FBPase through X-ray crystallography or cryo-electron microscopy, focusing on both apo and substrate/regulator-bound forms to identify unique binding pockets . Virtual screening of compound libraries against these structures can identify potential inhibitor scaffolds, with molecular dynamics simulations evaluating binding stability. Structure-activity relationship studies should guide iterative optimization of promising leads. Biochemical validation includes enzyme inhibition assays measuring IC₅₀ and K_i values, thermal shift assays to confirm binding, and isothermal titration calorimetry for thermodynamic characterization. Specificity must be assessed against mammalian FBPases to ensure selective targeting. Cellular assays evaluating inhibitor effects on bacterial growth, especially under gluconeogenic conditions, and host-pathogen interaction models provide functional validation. Finally, pharmacokinetic evaluation and in vivo efficacy studies in infection models complete the development pipeline.

How does the evolution of FBPase in E. faecalis compare to other enterococci and what are the implications for metabolic adaptation?

Evolutionary analysis of FBPase across enterococci species reveals significant insights into metabolic adaptation. E. faecalis FBPase class 3 likely evolved through selective pressures related to diverse carbon utilization needs across various host environments and infection sites. Comparative genomic analysis would involve constructing phylogenetic trees based on fbp sequences from multiple enterococci species, identifying conserved catalytic domains versus variable regulatory regions. Positive selection analysis (dN/dS ratios) could highlight residues under evolutionary pressure, particularly in regions involved in allosteric regulation. Horizontal gene transfer events might be detected through anomalous GC content, codon usage bias, or phylogenetic incongruence. The methodological approach should include ancestral sequence reconstruction to infer evolutionary trajectories and heterologous expression of ancestral and contemporary variants to compare kinetic parameters and regulatory responses. Implications for metabolic adaptation include understanding how different enterococci species evolved varying degrees of metabolic flexibility, potentially correlating with their ability to colonize different ecological niches or respond to host-imposed stresses during infection.

What role does E. faecalis FBPase play in biofilm formation and antibiotic resistance?

E. faecalis FBPase likely influences biofilm formation and antibiotic resistance through its central role in carbohydrate metabolism. Although the search results don't directly address this relationship, the interconnection between metabolism and virulence traits suggests important implications. Methodologically, researchers should investigate this question by creating FBPase deletion or conditional expression mutants and evaluating their biofilm forming capacity using crystal violet assays, confocal microscopy with fluorescent matrix stains, and scanning electron microscopy. Metabolomic profiling of wild-type versus FBPase-deficient biofilms would reveal carbohydrate composition differences, while transcriptomic comparisons could identify differentially expressed biofilm-related genes. For antibiotic resistance studies, minimum inhibitory concentration determinations, persister cell quantification, and antimicrobial tolerance assays under different carbon sources would establish connections between gluconeogenesis and resistance mechanisms. Time-lapse microscopy of biofilm development under metabolic stress or antibiotic exposure could further elucidate FBPase's role in adaptive responses. Mathematical modeling integrating metabolic flux data with biofilm architecture could provide predictive frameworks for therapeutic intervention targeting FBPase.

How does lactate production by E. faecalis affect its interactions with other microorganisms in polymicrobial infections?

Lactate production by E. faecalis, which is linked to its central carbon metabolism including the activity of FBPase, significantly impacts polymicrobial interactions. Research has shown that E. faecalis antagonizes Pseudomonas aeruginosa growth through mechanisms involving decreased environmental pH and lactate-mediated iron chelation . E. faecalis produces lactate through the action of lactate dehydrogenase (LDH), which catalyzes the reduction of pyruvate to lactate, and the ldh1 gene accounts for the majority of lactate produced . The increased production of lactate under iron-restricted conditions appears to be independent of the presence of other bacteria, suggesting it's an intrinsic metabolic response. This metabolic interaction exemplifies how E. faecalis can modulate the polymicrobial environment to its advantage. The methodology to further investigate this would include co-culture experiments with clinically relevant pathogens, metabolomic profiling of the shared growth medium, and genetic manipulation of FBPase and related metabolic enzymes to alter carbon flux through the lactate-producing pathway.

How can understanding E. faecalis FBPase function contribute to developing new antimicrobial strategies?

Understanding E. faecalis FBPase function opens several avenues for novel antimicrobial development. The unique regulatory mechanisms of bacterial FBPases, such as activation by phosphoenolpyruvate and sulfate rather than inhibition by AMP as in mammalian counterparts, provide a basis for selective targeting . Methodologically, researchers could develop specific inhibitors that exploit these differences, particularly targeting the allosteric activation sites that are conserved among heterotrophic bacteria but absent in mammalian FBPases. High-throughput screening approaches could identify compounds that disrupt FBPase function in bacterial systems without affecting the human enzyme. The fact that FBPase plays a role in bacterial survival within macrophages suggests that inhibiting this enzyme might attenuate E. faecalis virulence and enhance immune clearance . A combined approach using structure-based drug design informed by crystal structures, metabolic modeling to predict systemic effects of FBPase inhibition, and in vivo infection models would be required to translate fundamental knowledge into therapeutic applications.

What are the most promising techniques for studying conformational changes in E. faecalis FBPase during catalysis?

Recent advances in structural biology offer powerful tools for studying conformational changes in E. faecalis FBPase during catalysis. X-ray crystallography remains foundational, with time-resolved approaches capturing different catalytic states, as demonstrated in the remarkable study capturing over 1,000 snapshots of enzyme conformational changes . This approach would involve crystallizing E. faecalis FBPase in various states along the reaction coordinate, potentially using substrate or transition state analogs. Complementary to crystallography, hydrogen-deuterium exchange mass spectrometry (HDX-MS) can map conformational dynamics in solution, identifying regions with altered solvent accessibility during catalysis. Single-molecule Förster resonance energy transfer (smFRET) offers real-time visualization of enzyme movements when strategic residues are labeled with appropriate fluorophores. Molecular dynamics simulations, anchored by experimental structural data, can predict conformational trajectories and energy landscapes. Nuclear magnetic resonance (NMR) spectroscopy provides atomic-resolution information on protein dynamics in solution, particularly valuable for identifying allosteric networks connecting regulatory sites to catalytic centers. Integration of these complementary techniques would provide unprecedented insights into how E. faecalis FBPase structure enables its remarkable catalytic properties.

How might synthetic biology approaches be used to engineer E. faecalis FBPase for biotechnological applications?

Synthetic biology offers promising approaches for engineering E. faecalis FBPase for various biotechnological applications. Directed evolution strategies could enhance catalytic efficiency or stability through iterative rounds of mutagenesis and selection, potentially yielding enzymes with improved performance for industrial biocatalysis. Domain swapping between FBPases from different organisms could create chimeric enzymes with novel regulatory properties or substrate specificities. Site-directed mutagenesis targeting allosteric sites could generate variants responsive to non-natural regulators, enabling external control over gluconeogenic flux. From a methodological perspective, researchers would employ deep mutational scanning to comprehensively map sequence-function relationships, providing data for machine learning algorithms to predict beneficial mutations. Cell-free protein synthesis systems allow rapid prototyping of engineered variants before cellular implementation. Applications could include developing biosensors for metabolite detection, creating synthetic metabolic pathways for production of high-value chemicals, or engineering probiotics with enhanced survival capabilities under specific environmental conditions. Integration with systems biology approaches would ensure engineered enzymes function optimally within the context of cellular metabolism.