Recombinant Enterococcus faecalis Dephospho-CoA kinase (coaE)

What is the role of dephospho-CoA kinase (coaE) in Enterococcus faecalis metabolism?

Dephospho-CoA kinase (coaE) catalyzes the final step in coenzyme A biosynthesis, specifically the phosphorylation of the 3′-hydroxyl group of the ribose sugar moiety in dephosphocoenzyme A (dephospho-CoA) to form coenzyme A (CoA) . This reaction is critical for E. faecalis metabolism since CoA is an essential cofactor in numerous biochemical pathways. Approximately 4% of all enzymes use CoA or a thioester of CoA as a substrate, highlighting the significance of functional coaE in bacterial metabolism . In E. faecalis, this enzyme represents a potential target for antimicrobial development due to its essential role in cellular metabolism and potential differences from human homologues.

How is the coaE gene identified and characterized in E. faecalis?

The coaE gene in E. faecalis can be identified through sequence homology approaches similar to those used for other bacterial species. Initially, researchers purified the dephospho-CoA kinase protein from a related organism (Corynebacterium ammoniagenes) and performed N-terminal sequencing . This sequence data was used in BLAST searches to identify homologous proteins, including the yacE (later renamed coaE) gene product in E. coli, which showed 60% identity at the amino acid level . For E. faecalis, whole-genome sequencing data can be analyzed to identify the coaE homologue based on conserved motifs, particularly the Walker kinase motif found in positions 9-17 of the E. coli enzyme . Confirmation requires amplification of the gene by PCR, followed by cloning, expression, and functional verification through enzyme assays measuring ATP-dependent phosphorylation of dephospho-CoA.

What expression systems are optimal for producing recombinant E. faecalis coaE?

For recombinant E. faecalis coaE expression, E. coli BL21(DE3) with a pET vector system has proven effective for homologous enzymes . The protocol typically involves:

• PCR amplification of the E. faecalis coaE gene with appropriate restriction sites

• Insertion into an expression vector such as pET28b(+) with a His-tag for purification

• Transformation into E. coli BL21(DE3) cells

• Induction of protein expression using IPTG

• Cell lysis and initial purification using anion-exchange chromatography on DEAE Sepharose

• Further purification by anion-exchange chromatography on Q Sepharose

This approach typically yields pure enzyme as determined by SDS-PAGE, with expected molecular weight of approximately 22-25 kDa based on homologous proteins . Expression temperature optimization at 25-30°C may improve solubility compared to standard 37°C conditions.

What are the kinetic parameters of recombinant E. faecalis dephospho-CoA kinase?

While specific kinetic parameters for E. faecalis coaE are not directly reported in the provided search results, parameters can be extrapolated from homologous enzymes. The E. coli dephospho-CoA kinase has a Km for dephospho-CoA of 0.74 mM, which is higher than values reported for the mammalian enzymes (0.01 mM for rat liver enzyme) and somewhat higher than C. ammoniagenes dephospho-CoA kinase (0.12 mM) .

The kinetic characterization methodology for recombinant E. faecalis coaE would typically involve:

• Spectrophotometric assays coupling ADP formation to NADH oxidation via pyruvate kinase and lactate dehydrogenase

• Determination of optimal pH (likely alkaline, similar to rat liver enzyme)

• Measurement of activity across varying substrate concentrations (0.05-2.0 mM dephospho-CoA)

• Assessment of ATP dependence and metal ion requirements (typically Mg²⁺)

Researchers should anticipate a similar Km value to other bacterial coaE enzymes, probably in the 0.1-1.0 mM range for dephospho-CoA.

How does substrate specificity differentiate E. faecalis coaE from other homologues?

The substrate specificity of E. faecalis coaE likely mirrors that of other bacterial homologues, with highest activity toward dephospho-CoA. Based on data from related enzymes, E. faecalis coaE would be expected to phosphorylate alternate substrates like adenosine, AMP, and adenosine phosphosulfate (APS) at approximately 4-8% of the activity observed with dephospho-CoA .

To experimentally determine substrate specificity, researchers should:

• Prepare reaction mixtures containing purified enzyme, ATP, and various potential substrates

• Measure phosphorylation rates using either:

  • Coupled enzyme assays monitoring ADP formation

  • HPLC analysis of substrate consumption and product formation

  • ³¹P-NMR spectroscopy to verify 3′-hydroxyl phosphorylation

The enzyme likely exhibits high selectivity for the 3′-hydroxyl position, a rare specificity shared only with APS kinase among phosphoryl-transferring enzymes .

What purification strategies yield the highest activity for recombinant E. faecalis coaE?

Based on successful purification approaches for homologous enzymes, a multi-step purification strategy for recombinant E. faecalis coaE would include:

• Initial capture using anion-exchange chromatography on DEAE Sepharose

• Affinity chromatography:

  • If using His-tagged construct: Ni-NTA affinity chromatography

  • For native enzyme: Red A agarose with specific elution using ATP (as demonstrated for C. ammoniagenes enzyme)

• Further purification by anion-exchange chromatography on Q Sepharose

• Size exclusion chromatography for final polishing

This approach typically yields protein of >95% purity with specific activity preservation. A purification table similar to homologous enzymes might resemble:

How can structure-function relationships in E. faecalis coaE be investigated through site-directed mutagenesis?

Site-directed mutagenesis studies of E. faecalis coaE would primarily target conserved motifs identified through sequence alignment with homologues. The Walker kinase motif (corresponding to residues 9-17 in E. coli coaE) would be a primary target . A comprehensive mutagenesis strategy should include:

• Alanine scanning of the conserved Walker A motif (GXXGXGKT/S) essential for ATP binding

• Mutation of predicted catalytic residues involved in phosphoryl transfer

• Modification of residues in the dephospho-CoA binding pocket identified through homology modeling

• Analysis of structural elements that differentiate bacterial and mammalian enzymes

For each mutant:

• Express and purify using identical protocols to wild-type enzyme

• Determine kinetic parameters (kcat, Km) to assess effects on catalysis

• Evaluate structural integrity through circular dichroism spectroscopy

• When possible, obtain crystal structures to directly visualize changes in substrate binding

This approach would identify critical residues for potential inhibitor development and provide insights into the catalytic mechanism.

What is the relationship between E. faecalis coaE activity and pathogenicity?

The relationship between E. faecalis coaE and pathogenicity represents an important research direction. While specific data on E. faecalis coaE's role in pathogenicity is not directly presented in the search results, several investigative approaches could determine this relationship:

• Generate conditional coaE knockdown strains in E. faecalis and assess:

  • Growth rates under varying conditions

  • Biofilm formation capacity

  • Antibiotic susceptibility profiles

  • Virulence in infection models

• Analyze coaE expression patterns:

  • Compare expression between commensal and invasive isolates

  • Measure changes during transition from commensal to pathogenic states

  • Assess expression during bloodstream infection vs. intestinal colonization

Recent genome-wide association studies (GWAS) of E. faecalis isolates from different body sites suggest that infection by hospitalization status and body isolation source are heritable traits, with ~40% and ~30% of variation explained by E. faecalis genetics, respectively . This genetic basis of pathogenicity may involve multiple factors rather than individual loci, suggesting coaE might contribute to pathogenicity as part of broader metabolic adaptation during infection.

What approaches can identify potential inhibitors of E. faecalis coaE?

Developing inhibitors of E. faecalis coaE represents a promising antimicrobial strategy. A comprehensive inhibitor discovery workflow would include:

• High-throughput screening approaches:

  • Development of a fluorescence-based activity assay suitable for microplate format

  • Primary screening of compound libraries against purified recombinant enzyme

  • Counter-screening against mammalian dephospho-CoA kinase to identify selective inhibitors

• Structure-based design:

  • Homology modeling of E. faecalis coaE based on available bacterial structures

  • Virtual screening of compound libraries against the ATP-binding site and dephospho-CoA binding pocket

  • Fragment-based approaches to identify initial binding scaffolds

• Mechanism-based inhibitor design:

  • Synthesis of non-hydrolyzable ATP analogues

  • Development of transition-state mimetics for the phosphoryl transfer reaction

  • Creation of bisubstrate inhibitors linking ATP and dephospho-CoA structural elements

• Validation of candidate inhibitors:

  • IC₅₀ and Ki determination against purified enzyme

  • Assessment of antimicrobial activity against E. faecalis (MIC determination)

  • Cytotoxicity evaluation against mammalian cell lines

  • Confirmation of on-target activity through resistant mutant generation

How conserved is the coaE gene across different E. faecalis strains?

Analyzing coaE conservation across E. faecalis strains provides insight into its evolutionary importance. While the search results don't directly address this for E. faecalis specifically, comparative analyses of bacterial coaE genes revealed significant conservation across a wide range of organisms . For E. faecalis specifically, researchers should:

• Extract and align coaE sequences from multiple E. faecalis genome assemblies

• Calculate nucleotide and amino acid sequence identity percentages

• Identify conserved domains and variable regions

• Determine selective pressure by calculating dN/dS ratios across the gene

Such analysis would likely reveal high conservation of the catalytic core and ATP-binding motifs with possibly greater variation in peripheral regions. The recent GWAS study of 736 E. faecalis isolates indicates significant genetic variation across strains related to different infection contexts , though coaE specifically was not highlighted as highly variable.

How does E. faecalis coaE differ from mammalian dephospho-CoA kinase?

Understanding differences between bacterial and mammalian dephospho-CoA kinases is crucial for antimicrobial development. Key differences likely include:

• Structural organization:

  • Bacterial dephospho-CoA kinases, including E. faecalis coaE, are likely monofunctional enzymes of approximately 22-25 kDa

  • Mammalian equivalents are typically bifunctional enzymes with both phosphopantetheine adenylyltransferase and dephospho-CoA kinase activities organized in a larger protein (~57 kDa for pork liver enzyme)

• Substrate affinity:

  • Bacterial enzymes generally show higher Km values (0.12-0.74 mM) compared to mammalian enzymes (0.01 mM for rat liver)

  • This indicates potential differences in active site architecture that could be exploited for selective inhibitor design

• Sequence divergence:

  • While the search results note human homologues of monofunctional dephospho-CoA kinase , significant sequence differences likely exist

  • Detailed sequence alignment and structural comparison would reveal specific regions for selective targeting

This comparative analysis provides the foundation for developing antimicrobials with selectivity for bacterial enzymes over human counterparts.

What are the optimal storage conditions for maintaining recombinant E. faecalis coaE activity?

Maintaining enzyme stability during storage is critical for reliable experimental results. Based on protocols for similar enzymes, recommended storage conditions include:

• Short-term storage (1-2 weeks):

  • 4°C in buffer containing 50 mM Tris-HCl (pH 7.5-8.0), 100 mM NaCl, 5 mM MgCl₂, and 1 mM DTT

  • Addition of 10% glycerol to prevent protein aggregation

• Long-term storage:

  • Flash freezing aliquots in liquid nitrogen

  • Storage at -80°C in buffer containing 50 mM Tris-HCl (pH 7.5-8.0), 100 mM NaCl, 5 mM MgCl₂, 1 mM DTT, and 20% glycerol

  • Avoiding repeated freeze-thaw cycles

• Activity stabilization:

  • Addition of BSA (0.1 mg/mL) as a carrier protein

  • Inclusion of 0.1 mM ATP to stabilize the active site

• Stability assessment:

  • Regular activity testing using standard assay conditions

  • SDS-PAGE analysis to monitor potential degradation

The enzyme likely exhibits similar stability properties to the E. coli homologue, with activity retention for several months under optimal storage conditions.

How can the solubility and yield of recombinant E. faecalis coaE be optimized?

Optimizing solubility and yield of recombinant E. faecalis coaE requires attention to expression conditions and buffer composition. Based on successful approaches with similar enzymes:

• Expression optimization:

  • Lowering induction temperature to 16-25°C

  • Reducing IPTG concentration to 0.1-0.3 mM

  • Extending expression time to 16-20 hours

  • Co-expression with chaperones (GroEL/GroES system) if inclusion body formation occurs

• Lysis buffer optimization:

  • Use of 50 mM Tris-HCl (pH 7.5-8.0), 300 mM NaCl, 5 mM MgCl₂, 1 mM DTT

  • Addition of 10% glycerol and 0.1% nonionic detergent (e.g., Triton X-100)

  • Inclusion of protease inhibitors to prevent degradation

• Solubility enhancers during purification:

  • Maintaining 5-10% glycerol throughout purification

  • Addition of 50-100 mM arginine to prevent aggregation

  • Use of stabilizing ligands (0.1 mM ATP) during purification steps

• Protein refolding if necessary:

  • Gradual dialysis from denaturing conditions (6 M urea or 8 M guanidine-HCl)

  • On-column refolding during affinity purification

These approaches typically increase soluble protein yield from <10 mg/L to >50 mg/L of bacterial culture.

How might research on E. faecalis coaE contribute to understanding antibiotic resistance mechanisms?

Investigating E. faecalis coaE in the context of antibiotic resistance offers several promising research directions:

• Metabolic adaptation during antibiotic exposure:

  • Analysis of coaE expression levels in response to different antibiotics

  • Determination if increased CoA biosynthesis contributes to stress responses during antibiotic treatment

  • Assessment of CoA-dependent detoxification pathways activated during antibiotic challenge

• Relationship to antibiotic resistance profiles:

  • Comparison of coaE sequence and expression between antibiotic-susceptible and resistant isolates

  • Investigation of potential coaE regulation by resistance-associated transcription factors

  • The GWAS study of E. faecalis isolates indicates that antibiotic resistance patterns, rather than individual genetic variations, significantly influence infection characteristics

• Metabolic burden of resistance mechanisms:

  • Evaluation of how acquisition of resistance determinants affects CoA homeostasis

  • Investigation of whether coaE upregulation compensates for metabolic costs of resistance

• Biofilm formation and persistence:

  • Determination of coaE's role in biofilm-associated antibiotic tolerance

  • Assessment of CoA-dependent signaling in persister cell formation

This research direction could identify metabolic vulnerabilities in resistant E. faecalis strains that might be exploited therapeutically.

What aspects of coaE function in E. faecalis require further investigation?

Despite the significant progress in understanding bacterial dephospho-CoA kinases, several aspects of E. faecalis coaE function warrant further investigation:

• Structural biology:

  • Determination of crystal structure in different functional states (apo, ATP-bound, dephospho-CoA-bound)

  • Elucidation of conformational changes during catalysis

• Regulation mechanisms:

  • Identification of transcriptional and post-translational regulation

  • Assessment of allosteric regulation by metabolites

  • Investigation of genetic context and potential operon structure

• Role in specialized metabolic contexts:

  • Function during biofilm formation

  • Activity changes during transition from commensal to pathogenic states

  • Contribution to stress responses and adaptation to host environments

  • Analysis of potential moonlighting functions beyond CoA biosynthesis

• Interaction networks:

  • Identification of protein-protein interactions

  • Integration with other metabolic pathways

  • Potential coordination with other CoA biosynthetic enzymes

These research directions would significantly advance understanding of E. faecalis metabolism and potentially reveal new therapeutic approaches against this opportunistic pathogen.