Recombinant Enterococcus faecalis Deoxyribose-phosphate aldolase (deoC)

What is Deoxyribose-phosphate aldolase (deoC) in Enterococcus faecalis?

Deoxyribose-phosphate aldolase (deoC) in Enterococcus faecalis is an enzyme belonging to the DERA family involved in the utilization of extracellular deoxyribonucleotides as energy sources . It catalyzes the reversible transformation of d-2-deoxyribose-5-phosphate (DRP) into d-glyceraldehyde-3-phosphate and acetaldehyde, which subsequently enter glycolysis and the Krebs cycle, respectively . Like other DERAs, the E. faecalis enzyme is unique among aldolases because it can catalyze aldol condensation reactions between two aldehydes, whereas other aldolases typically use ketones as aldol donors and aldehydes as acceptors . The enzyme is part of the core genome machinery in E. faecalis and likely shares structural similarities with characterized DERAs from other bacterial species, featuring the conserved catalytic lysine residue essential for forming a Schiff base intermediate during the reaction mechanism.

How does E. faecalis deoC compare structurally to other bacterial DERAs?

While the specific crystal structure of E. faecalis deoC has not been fully characterized in the provided search results, comparative analysis with other bacterial DERAs suggests it likely adopts the ubiquitous triosephosphate isomerase (TIM) barrel (α/β)8 fold . This structural arrangement is consistent with other bacterial DERAs, such as those from E. coli, B. halodurans (BH1352), and T. maritima (TM1559) . A distinguishing feature would be the position and accessibility of the catalytic lysine residue, which forms the Schiff base with substrates. The C-terminal tyrosine residue, which has been shown to be crucial for enzyme activity in E. coli DeoC (Tyr 259), is likely also present in the E. faecalis enzyme . Recent structural studies of BH1352 and TM1559 revealed the first full-length DERA structures showing this C-terminal tyrosine (Tyr 224 in BH1352 and Tyr 246 in TM1559), with different positioning - on a flexible strand in BH1352 and on a C-terminal α-helix in TM1559 . This structural diversity suggests E. faecalis deoC might have unique structural features that influence its catalytic properties.

What biochemical reactions does E. faecalis deoC catalyze?

E. faecalis deoC catalyzes two primary reactions:

• Retro-aldol Cleavage: The enzyme cleaves d-2-deoxyribose-5-phosphate (DRP) into d-glyceraldehyde-3-phosphate and acetaldehyde . This reaction is part of the catabolic pathway for deoxyribonucleotides.

• Aldol Condensation: In the reverse reaction, deoC catalyzes the aldol condensation between acetaldehyde (the donor molecule) and d-glyceraldehyde-3-phosphate (the acceptor molecule) to produce DRP . This reaction proceeds through a covalent Schiff base intermediate formed between the catalytic lysine residue and acetaldehyde.

Additionally, like other DERAs, E. faecalis deoC likely has the capability to catalyze sequential aldol reactions, potentially ligating multiple aldehyde molecules in a stereoselective manner . This property makes DERAs valuable biocatalysts for synthetic organic chemistry applications, including the production of compounds like 1,3-butanediol through the condensation of two acetaldehyde molecules followed by reduction .

How can E. faecalis deoC be optimized for improved catalytic efficiency in biocatalysis applications?

Optimization of E. faecalis deoC for enhanced catalytic efficiency would benefit from structure-guided protein engineering approaches similar to those applied to other DERAs. Based on studies with BH1352 from B. halodurans, targeted mutations of active site residues can significantly impact catalytic properties . For instance, mutations equivalent to the F160Y and F160Y/M173I variants in BH1352, which showed 2.5-fold increases in acetaldehyde transformation to 1,3-butanediol, could be explored in E. faecalis deoC . The optimization process should include:

• Structure determination: Solving the crystal structure of E. faecalis deoC to identify key catalytic residues and substrate binding sites.

• Site-directed mutagenesis: Targeting conserved residues known to influence substrate specificity and catalytic efficiency in other DERAs.

• High-throughput screening: Developing efficient assays to evaluate variant libraries for improved activity with specific substrates.

• pH optimization: Given that some DERAs like BH1352 from the alkaliphilic B. halodurans show broad pH optima (7.2-9.5), exploring the pH profile of E. faecalis deoC could reveal conditions for maximal activity .

• Stability engineering: Enhancing resistance to aldehyde-induced inactivation, a common limitation for DERAs in industrial applications.

These approaches could yield E. faecalis deoC variants with enhanced catalytic properties for specific biocatalytic applications, particularly for stereoselective aldol condensations.

What role might E. faecalis deoC play in biofilm formation and pathogenicity?

While the provided search results don't directly connect E. faecalis deoC to biofilm formation, broader genomic studies of E. faecalis indicate multiple genetic determinants involved in biofilm formation . Biofilm formation plays a major role in the pathogenesis of opportunistic antibiotic-resistant E. faecalis infections and in the transfer of antibiotic resistance genes . Considering deoC's role in deoxyribonucleotide metabolism, it could potentially contribute to biofilm dynamics through several mechanisms:

• Nucleotide recycling: By metabolizing extracellular DNA (a key component of the biofilm matrix), deoC might help regulate biofilm structure and provide carbon sources during biofilm growth.

• Stress response: Altered deoC expression might occur during the transition to biofilm growth, potentially as part of the adaptation to nutrient limitation.

• Metabolic adaptation: The enzyme's activity could contribute to the distinct metabolic state of biofilm cells compared to planktonic cells.

Further research using techniques like recombinase in vivo expression technology (RIVET), which has identified 68 genetic loci involved in biofilm formation, might reveal connections between deoC activity and E. faecalis biofilm development . Integration of transcriptomic and metabolomic approaches would be valuable to clarify any relationship between deoxyribose metabolism and biofilm-associated phenotypes.

How does the kinetic profile of E. faecalis deoC compare with DERAs from other organisms?

A comprehensive kinetic comparison would require experimental determination of E. faecalis deoC parameters, but insights can be drawn from studies of other bacterial DERAs:

E. faecalis deoC would likely exhibit kinetic parameters within the range of these characterized DERAs . Given that E. faecalis is highly adaptable to various environments, its deoC might have evolved distinct substrate affinities or catalytic efficiencies compared to other bacterial enzymes. Of particular interest would be its performance in acetaldehyde condensation reactions, which varies significantly among DERAs and has important implications for biocatalytic applications . The enzyme's stability and activity under various pH conditions, temperature ranges, and in the presence of potential inhibitors would further distinguish its kinetic profile from other bacterial DERAs.

What are the optimal expression and purification strategies for recombinant E. faecalis deoC?

Based on successful approaches with other bacterial DERAs, the following expression and purification strategy is recommended for recombinant E. faecalis deoC:

Expression System:

• Vector selection: pET-based vectors with T7 promoter systems provide high-level expression for bacterial proteins .

• Host strain: E. coli BL21(DE3) or derivatives like Rosetta for potential rare codon usage.

• Fusion tags: N-terminal 6×His-tag facilitates purification while minimally affecting enzyme activity.

• Induction conditions: IPTG concentration of 0.1-0.5 mM at lower temperatures (16-25°C) for 16-20 hours can improve soluble protein yield.

Purification Protocol:

• Cell lysis: Sonication or mechanical disruption in a buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10% glycerol, and 1 mM DTT.

• Initial purification: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin.

• Secondary purification: Size exclusion chromatography for higher purity, particularly important for crystallography studies.

• Tag removal: If necessary, incorporate a TEV protease cleavage site between the tag and protein.

Quality Control:

• SDS-PAGE to assess purity

• Activity assays using the coupled DRP cleavage reaction system with glyceraldehyde-3-phosphate dehydrogenase and triosephosphate isomerase

• Thermal shift assays to evaluate protein stability

This approach should yield enzymatically active E. faecalis deoC suitable for biochemical characterization and crystallization attempts, as has been demonstrated for other bacterial DERAs .

How can researchers accurately measure and characterize the catalytic activities of E. faecalis deoC?

Comprehensive characterization of E. faecalis deoC catalytic activities requires multiple complementary assays:

1. Retro-aldol DRP Cleavage Activity:

• Coupled enzyme assay: Link DRP cleavage to NADH formation via glyceraldehyde-3-phosphate dehydrogenase and triosephosphate isomerase, monitoring absorbance at 340 nm .

• Direct product quantification: Measure acetaldehyde formation using aldehyde dehydrogenase or gas chromatography.

2. Aldol Condensation Activity:

• Direct product analysis: Use HPLC or LC-MS to detect and quantify DRP formation from acetaldehyde and d-glyceraldehyde-3-phosphate.

• Coupled enzyme system: For acetaldehyde condensation, couple with an aldo-keto reductase (e.g., PA1127 from P. aeruginosa) to convert the intermediate 3-hydroxybutanal to 1,3-butanediol, which can be quantified by GC-MS .

3. Kinetic Parameter Determination:

• Measure initial reaction rates at varying substrate concentrations

• Plot data using Michaelis-Menten or appropriate enzyme kinetics models

• Calculate Km, Vmax, kcat, and catalytic efficiency (kcat/Km)

4. pH, Temperature, and Stability Profiling:

• Determine optimal pH range using buffers from pH 5.0-10.0

• Establish temperature optimum and stability profile

• Assess aldehyde tolerance by pre-incubation with varying concentrations of acetaldehyde

5. Substrate Specificity Analysis:

• Test various aldehydes as potential substrates

• Evaluate stereospecificity using chiral analysis of products

These methods, which have been successfully applied to other DERAs, provide a comprehensive toolkit for characterizing the catalytic properties of E. faecalis deoC and comparing it with enzymes from other organisms .

What crystallization strategies are most promising for determining the structure of E. faecalis deoC?

Based on successful crystallization of related DERAs, the following strategies are recommended for determining the structure of E. faecalis deoC:

1. Protein Preparation:

• Purify to >95% homogeneity using affinity chromatography followed by size exclusion

• Verify monodispersity by dynamic light scattering

• Test both tagged and untagged versions of the protein

• Concentrate to 10-20 mg/ml in a buffer containing 20 mM Tris-HCl (pH 7.5-8.0), 150 mM NaCl

2. Crystallization Screening:

• Employ sitting-drop vapor diffusion method, which successfully yielded crystals for BH1352 and TM1559

• Use commercial sparse matrix screens (Hampton, Molecular Dimensions, Qiagen)

• Test protein:reservoir ratios of 1:1, 1:2, and 2:1

• Incubate at both 4°C and 20°C

3. Optimization Strategies:

• Fine-tune promising conditions by varying precipitant concentration, pH, and additives

• Try seeding techniques to improve crystal quality

• Consider co-crystallization with substrates or substrate analogs to capture different conformational states

• Test surface entropy reduction mutations if initial crystallization attempts fail

4. Diffraction and Structure Determination:

• Cryoprotect crystals using reservoir solution supplemented with 20-25% glycerol or ethylene glycol

• Collect data at synchrotron radiation sources for highest resolution

• Use molecular replacement with known DERA structures (BH1352, TM1559, or E. coli DeoC) as search models

• Pay special attention to the C-terminal region containing the catalytically important tyrosine residue, which is often flexible and difficult to resolve

These approaches mirror the successful crystallization strategies that yielded high-resolution structures (1.40–2.50 Å) for BH1352 and TM1559, which represent the first full-length DERA structures revealing the presence of the C-terminal tyrosine residue .

How might E. faecalis deoC be utilized in biocatalytic synthesis of valuable compounds?

E. faecalis deoC holds significant potential for biocatalytic applications, particularly in the stereoselective synthesis of valuable compounds. Based on the capabilities of characterized DERAs, potential applications include:

1. Production of (R)-1,3-butanediol:

• E. faecalis deoC could catalyze the condensation of two acetaldehyde molecules to form 3-hydroxybutanal, which can then be reduced to (R)-1,3-butanediol by aldo-keto reductases .

• This pathway represents a valuable alternative to traditional chemical synthesis, offering stereoselectivity and milder reaction conditions.

2. Synthesis of statin side chains:

• DERAs can catalyze sequential aldol reactions involving chloroacetaldehyde and acetaldehyde to form precursors for statin side chains .

• E. faecalis deoC might offer unique substrate specificity or stability advantages for these reactions.

3. Production of unnatural deoxyribose derivatives:

• By accepting various aldehyde substrates, E. faecalis deoC could potentially generate modified deoxysugar structures for pharmaceutical applications.

4. Multi-enzyme cascade reactions:

• Integration with other enzymes could enable one-pot synthesis of complex chiral compounds from simple starting materials.

Implementation would require characterization of E. faecalis deoC's substrate scope, optimization of reaction conditions, and potentially protein engineering to enhance stability and activity with non-natural substrates. The enzyme could be deployed either as purified protein in cell-free systems or through whole-cell biocatalysis using engineered E. coli strains expressing E. faecalis deoC .

What are the key challenges in engineering E. faecalis deoC for enhanced industrial applications?

Engineering E. faecalis deoC for industrial applications faces several significant challenges:

1. Aldehyde toxicity and enzyme inhibition:

• Aldehydes, both substrates and products, can inactivate enzymes through irreversible adduct formation with nucleophilic residues.

• Engineering strategies must focus on enhancing aldehyde tolerance while maintaining catalytic efficiency.

2. Substrate specificity:

• Modifying the enzyme to accept non-natural substrates while maintaining stereocontrol requires precise manipulation of the active site architecture.

• Crystal structures of DERAs reveal that substrate recognition involves multiple residues working in concert .

3. Reaction equilibrium:

• The reversible nature of aldol reactions can limit product yields.

• Coupling with additional reactions to remove products or optimize reaction conditions may be necessary.

4. Scalability and stability:

• Industrial applications require enzymes that maintain activity under process conditions.

• Thermal stability, pH tolerance, and long-term operational stability must be optimized.

5. Protein expression levels:

• High-level expression of active enzyme is critical for economic feasibility.

• Codon optimization, signal peptide engineering, and fermentation optimization may be required.

Addressing these challenges would benefit from integrated approaches combining structural biology, computational design, directed evolution, and process engineering. The successful engineering of BH1352 variants with improved activity for 1,3-butanediol production demonstrates the feasibility of enhancing DERA performance through targeted mutations . Similar strategies focusing on key active site residues could be applied to E. faecalis deoC to overcome these limitations and expand its industrial utility.

How does research on E. faecalis deoC contribute to understanding the evolution of substrate specificity in the DERA enzyme family?

Research on E. faecalis deoC provides valuable insights into the evolution of substrate specificity across the DERA enzyme family:

1. Phylogenetic context:

• Comprehensive phylogenetic analysis has revealed five major clusters of DERA proteins, including bacterial domain, Firmicutes (Bacilli and Clostridia), Proteobacteria, and mixed clusters .

• Positioning E. faecalis deoC within this evolutionary framework helps identify lineage-specific adaptations that influence substrate preferences.

2. Structure-function relationships:

• Comparing the structural features of E. faecalis deoC with other DERAs reveals how subtle variations in active site architecture translate to differences in substrate recognition.

• The positioning of the C-terminal tyrosine residue, which varies between different DERAs (flexible strand in BH1352 versus C-terminal α-helix in TM1559), may reflect evolutionary adaptations to different substrate profiles .

3. Host organism adaptation:

• E. faecalis inhabits diverse ecological niches, from the human gut to environmental sources, suggesting its deoC may have evolved specialized features for functioning under varied conditions.

• Studying how these adaptations correlate with enzyme properties provides insights into evolutionary pressures shaping substrate specificity.

4. Catalytic promiscuity:

• DERAs exhibit varying degrees of activity with different aldehyde substrates, reflecting their evolutionary history.

• Characterizing the substrate range of E. faecalis deoC contributes to understanding how catalytic promiscuity evolves and can be harnessed for biotechnological applications.

This research not only enhances our fundamental understanding of enzyme evolution but also provides practical guidance for engineering DERAs with novel specificities. The conservation patterns across bacterial DERAs suggest that insights from E. faecalis deoC could be broadly applicable to understanding and manipulating this important enzyme family .

What strategies can resolve expression and solubility issues with recombinant E. faecalis deoC?

When encountering expression and solubility challenges with recombinant E. faecalis deoC, researchers should implement the following systematic troubleshooting approaches:

1. Expression optimization:

• Temperature modulation: Lower induction temperatures (16-20°C) often improve folding and solubility.

• Inducer concentration: Test IPTG concentrations from 0.1-1.0 mM to find optimal expression conditions.

• Expression duration: Shorter induction times may reduce inclusion body formation.

• Media formulation: Enhanced media (e.g., Terrific Broth) or supplementation with osmolytes like sorbitol can improve soluble yields.

2. Fusion tag strategies:

• Solubility enhancers: Test MBP, SUMO, or Thioredoxin fusion tags known to enhance solubility.

• Tag position: Compare N-terminal versus C-terminal tag placement, considering the critical role of the C-terminal tyrosine in DERA activity .

• Dual tagging: Combining affinity and solubility tags can sometimes overcome expression issues.

3. Host strain selection:

• Codon optimization: E. faecalis has different codon usage than E. coli; use strains like Rosetta that supply rare tRNAs.

• Chaperone co-expression: Strains that co-express folding chaperones (GroEL/ES, DnaK) can improve folding.

• Specialized strains: C41(DE3) or C43(DE3) are designed for toxic or membrane protein expression.

4. Protein extraction optimization:

• Lysis buffer screening: Test various buffers with different pH values, salt concentrations, and additives.

• Stabilizing additives: Include glycerol (10-20%), reducing agents, and specific cofactors or substrates.

• Extraction methods: Compare sonication, French press, and chemical lysis for optimal protein release.

5. Refolding approaches:

• If inclusion bodies persist, develop a refolding protocol using gradual dialysis or on-column refolding.

• Include stabilizing agents during refolding to prevent aggregation.

These strategies have proven effective for expressing challenging bacterial enzymes and should address most solubility issues with recombinant E. faecalis deoC .

How can researchers distinguish between E. faecalis deoC activity and endogenous E. coli DeoC in experimental systems?

Differentiating E. faecalis deoC activity from endogenous E. coli DeoC in experimental systems requires strategic approaches:

1. Genetic knockout strategies:

• Use E. coli expression strains with deoC gene deletions (ΔdeoC) to eliminate background activity.

• Complement these strains with the E. faecalis deoC gene to ensure observed activity comes solely from the recombinant enzyme.

2. Biochemical differentiation:

• Kinetic parameters: Determine and compare substrate affinities (Km) and catalytic efficiencies (kcat/Km) between the purified enzymes.

• pH profiles: Establish distinct pH optima that allow selective measurement of E. faecalis deoC activity.

• Substrate specificity: Identify differential activities toward non-natural substrates that might be preferentially processed by one enzyme over the other.

3. Immunological approaches:

• Develop antibodies specific to E. faecalis deoC that don't cross-react with E. coli DeoC.

• Use these for Western blotting, enzyme-linked immunosorbent assays (ELISA), or immunoprecipitation to quantify specific protein levels.

4. Tagging and purification strategies:

• Express E. faecalis deoC with affinity tags that allow complete separation from the native E. coli enzyme.

• Verify purification efficacy using mass spectrometry to confirm protein identity.

5. Selective inhibition:

• Identify inhibitors with differential effects on the two enzymes.

• Use these compounds to selectively suppress E. coli DeoC activity when measuring E. faecalis deoC function.

These approaches ensure accurate attribution of observed enzymatic activities, particularly important when studying subtle differences in catalytic properties between homologous enzymes from different bacterial species .

What are the common pitfalls in kinetic characterization of E. faecalis deoC and how can they be avoided?

Kinetic characterization of E. faecalis deoC presents several potential pitfalls that researchers should anticipate and address:

1. Product inhibition effects:

• Pitfall: Acetaldehyde and other aldehyde products can inhibit DERA activity, leading to non-linear kinetics and underestimated reaction rates.

• Solution: Design assays with continuous product removal or use coupled enzyme systems that immediately convert products to non-inhibitory compounds . Monitor reaction progress over shorter time intervals to minimize product accumulation.

2. Substrate stability issues:

• Pitfall: Aldehyde substrates are prone to oxidation, polymerization, and hydration in aqueous solutions.

• Solution: Prepare fresh substrate solutions immediately before assays. Use anaerobic conditions when possible and include reducing agents in assay buffers. Verify actual substrate concentrations spectrophotometrically before each experiment.

3. Coupled assay interferences:

• Pitfall: Components in coupled enzyme assays may affect E. faecalis deoC activity directly.

• Solution: Validate that coupling enzymes are not rate-limiting by using excess amounts. Perform control experiments with varying concentrations of coupling enzymes to ensure observed rates reflect deoC activity .

4. Buffer and pH effects:

• Pitfall: DERA activity is highly pH-dependent, and buffer components can influence activity.

• Solution: Characterize the enzyme across a wide pH range (5.0-10.0) with overlapping buffer systems to identify optimal conditions . Test multiple buffer types at the same pH to identify any specific buffer effects.

5. Data analysis complexities:

• Pitfall: Some DERAs exhibit non-Michaelis-Menten kinetics, particularly at high substrate concentrations.

• Solution: Apply appropriate kinetic models that account for substrate inhibition, cooperativity, or other non-classical behaviors. Collect sufficient data points, particularly at low substrate concentrations, to accurately determine initial kinetic parameters.

By anticipating these challenges and implementing appropriate experimental designs, researchers can generate reliable kinetic data for E. faecalis deoC that facilitates meaningful comparisons with other DERAs .

What are the most promising future research directions for E. faecalis deoC?

The study of E. faecalis deoC offers several compelling research directions that could significantly advance both fundamental understanding and practical applications:

1. Structural biology:

• Determination of the high-resolution crystal structure of E. faecalis deoC, particularly capturing the position of the catalytically important C-terminal tyrosine residue .

• Co-crystallization with substrates and reaction intermediates to elucidate the molecular basis of catalysis and substrate recognition.

2. Enzyme engineering:

• Rational design of E. faecalis deoC variants with enhanced stability, broader substrate scope, or improved stereoselectivity based on insights from successful engineering of other DERAs like BH1352 .

• Directed evolution approaches targeting industrial applications, particularly the synthesis of pharmaceutical precursors.

3. Metabolic context:

• Investigation of the physiological role of deoC in E. faecalis metabolism, particularly during colonization of different host niches.

• Exploration of potential connections between deoxyribose metabolism and biofilm formation, which plays a major role in E. faecalis pathogenesis .

4. Biocatalytic applications:

• Development of optimized biocatalytic processes using E. faecalis deoC for the production of chiral building blocks and pharmaceutical intermediates.

• Integration into multi-enzyme cascades for the synthesis of complex molecules from simple, renewable starting materials.

5. Comparative enzymology:

• Comprehensive comparison of E. faecalis deoC with DERAs from diverse bacterial sources to understand the molecular basis of their different substrate specificities and catalytic efficiencies .

• Investigation of how evolutionary adaptations to different ecological niches have shaped DERA function across bacterial species.

These research directions would not only enhance our understanding of this industrially relevant enzyme but could also lead to valuable biotechnological applications and insights into E. faecalis biology.

What are the key unresolved questions about E. faecalis deoC that merit further investigation?

Despite advances in DERA research, several critical questions about E. faecalis deoC remain unresolved and warrant dedicated investigation:

1. Structural determinants of activity:

• How does the structure of E. faecalis deoC compare with well-characterized DERAs, particularly regarding the position and dynamics of the C-terminal tyrosine residue that plays a crucial role in catalysis ?

• What structural features determine substrate specificity and catalytic efficiency in E. faecalis deoC compared to other bacterial DERAs?

2. Physiological significance:

• What is the precise role of deoC in E. faecalis metabolism during colonization, infection, and biofilm formation ?

• How is deoC expression regulated in response to environmental conditions, and does this regulation differ from other bacteria?

3. Catalytic mechanism nuances:

• Does E. faecalis deoC utilize the same proton relay system involving aspartate and lysine residues as documented in E. coli DeoC ?

• Are there unique aspects of the catalytic mechanism that could be exploited for inhibitor design or enzyme engineering?

4. Performance characteristics:

• How does E. faecalis deoC compare with other DERAs in terms of aldehyde tolerance, stability, and efficiency in biocatalytic applications ?

• What are the kinetic parameters of E. faecalis deoC with various natural and non-natural substrates?

5. Evolutionary context:

• How has the deoC gene evolved in E. faecalis compared to other enterococci and more distant bacterial species?

• Are there strain-specific variations in deoC sequence and activity among clinical and environmental E. faecalis isolates?

Addressing these questions would fill significant knowledge gaps about E. faecalis deoC and potentially reveal new applications or therapeutic targets related to this enzyme.

How might research on E. faecalis deoC contribute to addressing antibiotic resistance and biofilm-related infections?

Research on E. faecalis deoC could provide novel insights and strategies for addressing the pressing challenges of antibiotic resistance and biofilm-related infections:

1. Novel therapeutic targets:

• If deoC plays an essential role in E. faecalis metabolism during infection or biofilm formation, it could represent a novel drug target .

• Inhibitors of deoC might disrupt nucleotide salvage pathways critical for bacterial survival in certain infection sites.

2. Biofilm disruption strategies:

• Understanding the potential involvement of deoC in biofilm formation could reveal new approaches to disrupt these structures .

• Manipulating deoxyribose metabolism might affect extracellular DNA processing, an important component of biofilm matrices.

3. Metabolic vulnerabilities:

• Characterization of E. faecalis deoC might uncover metabolic vulnerabilities that could be exploited by combination therapies.

• Targeting pathways connected to deoxyribose metabolism could enhance the efficacy of existing antibiotics.

4. Diagnostic applications:

• If deoC expression is differentially regulated during biofilm formation, it could serve as a biomarker for biofilm-associated infections .

• Developing diagnostic tools targeting deoC expression could help identify E. faecalis biofilm infections.

5. Anti-virulence approaches:

• Rather than killing bacteria directly, disrupting metabolic pathways involving deoC could potentially reduce virulence without imposing strong selective pressure for resistance.

• This approach aligns with current strategies to develop anti-virulence therapies as alternatives to conventional antibiotics.