Recombinant Enterococcus faecalis Ferredoxin--NADP reductase (EF_2899)

What is the basic function of Ferredoxin--NADP Reductase in Enterococcus faecalis?

Ferredoxin--NADP Reductase (FNR) in Enterococcus faecalis is an FAD-containing enzyme that catalyzes the transfer of electrons from ferredoxin (Fd) to NADP+ to generate NADPH. Unlike its plant counterparts that function in photosynthesis, the bacterial FNR (including EF_2899) plays crucial roles in redox metabolism and electron transfer processes essential for bacterial survival under various environmental conditions .

To study this function experimentally, researchers should employ spectrophotometric assays measuring NADPH formation at 340 nm when the purified enzyme is incubated with ferredoxin and NADP+. The reaction should be monitored in appropriate buffer conditions (typically 50 mM Tris-HCl, pH 7.5) with temperature control (30-37°C). When designing these experiments, it's essential to include proper controls to account for non-enzymatic reduction and to validate enzyme activity through kinetic analyses.

How does the structure of E. faecalis FNR compare to other bacterial FNRs?

For structural analysis, researchers should consider:

• Obtaining high-resolution crystal structures (≤1.5 Å resolution)

• Performing comparative analyses with structurally resolved FNRs from other organisms

• Examining active site architecture, particularly the nicotinamide-flavin interaction

• Analyzing patterns of amino acid conservation among bacterial FNRs

The structural data for related FNRs is available in the PDB database, with multiple variant structures such as wild-type (3LO8) and several Y316 mutants including Y316S, Y316A, and Y316F in different binding states with nicotinamide, NADP+, or NADPH .

What are the optimal conditions for expressing recombinant EF_2899 in E. coli?

For optimal expression of recombinant EF_2899 in E. coli, researchers should implement a systematic approach addressing multiple variables:

• Expression vector selection: pET-based vectors with T7 promoters typically yield high expression levels. The EF_2899 gene should be codon-optimized for E. coli expression.

• Host strain selection: BL21(DE3) or Rosetta(DE3) strains are recommended, with the latter being particularly useful if EF_2899 contains rare codons.

• Induction parameters:

  • Temperature: Lower temperatures (16-20°C) often improve protein solubility

  • IPTG concentration: 0.1-0.5 mM typically suffices

  • Induction timing: Mid-log phase (OD600 0.6-0.8) generally yields optimal results

  • Duration: 16-18 hours at lower temperatures or 3-4 hours at 37°C

• Culture media: Enriched media such as Terrific Broth can enhance yield. For isotopic labeling studies, minimal media with appropriate supplements should be used.

• Additives: Consider adding 0.1 mM FAD to the culture medium to facilitate cofactor incorporation during expression.

When troubleshooting expression issues, employ SDS-PAGE and Western blotting to assess protein production, and vary induction parameters systematically to identify optimal conditions for soluble protein production .

What purification strategy yields the highest purity and activity for recombinant EF_2899?

An effective multi-step purification strategy for recombinant EF_2899 involves:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin if the protein carries a His-tag. Equilibrate column with 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole; wash with increasing imidazole concentrations (20-40 mM); elute with 250 mM imidazole.

• Intermediate purification: Ion exchange chromatography (IEX) using Q-Sepharose. Use a linear NaCl gradient (0-500 mM) in 50 mM Tris-HCl pH 8.0 buffer.

• Polishing step: Size exclusion chromatography using Superdex 75 or 200 in 25 mM Tris-HCl pH 7.5, 150 mM NaCl.

• Buffer optimization: The final storage buffer should contain 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT, 10% glycerol, and potentially 10 μM FAD to ensure cofactor saturation.

For quality control, implement:

• SDS-PAGE (>95% purity)

• UV-visible spectroscopy (characteristic FAD spectrum with A₂₇₅/A₄₅₀ ratio)

• Enzymatic activity assays (specific activity >10 μmol NADPH formed/min/mg protein)

• Mass spectrometry to confirm protein identity

This approach typically yields 15-20 mg of pure, active enzyme per liter of bacterial culture .

What experimental design strategies should be employed when studying EF_2899 kinetics?

When designing experiments to study EF_2899 kinetics, researchers should employ a systematic approach:

• Initial rate determination: Ensure measurements are made in the linear range of the reaction (typically <10% substrate conversion) to accurately determine initial velocities.

• Substrate concentration ranges:

  • For NADP+: 10-500 μM

  • For NADPH: 10-500 μM

  • For ferredoxin: 1-100 μM

  • Test at minimum 7-8 concentrations across these ranges

• Experimental conditions optimization:

  • Buffer composition: Test multiple buffers (HEPES, Tris, phosphate) at pH 6.5-8.0

  • Temperature dependence: Collect data at 25°C, 30°C, and 37°C

  • Ionic strength effects: Vary NaCl concentration (0-300 mM)

• Data analysis approach:

  • Fit data to appropriate models (Michaelis-Menten, Hill, etc.)

  • Use nonlinear regression, not linear transformations

  • Perform statistical validation of fitted parameters

  • Consider global fitting approaches for complex mechanisms

• Controls and validation:

  • Include enzyme-free controls

  • Perform replicate measurements (minimum triplicate)

  • Validate with alternative assay methods

The experimental design should account for potential product inhibition and substrate depletion. Researchers should analyze data comprehensively using statistical approaches to define confidence intervals for kinetic parameters .

How do mutations at the conserved Tyr316 residue affect the hydride transfer mechanism of FNR?

Mutations at the conserved Tyr316 residue (equivalent position in E. faecalis FNR) significantly impact the hydride transfer mechanism between nicotinamide and flavin. High-resolution crystallographic studies (~1.5 Å) of corn root FNR variants (Tyr316Ser, Tyr316Ala, and Tyr316Phe) reveal that:

• Structural changes:

  • Y316S and Y316A mutations alter the positioning of the nicotinamide ring relative to the flavin, affecting the critical N5-C4 distance for hydride transfer

  • These variants show distinct patterns of FAD covalent distortion compared to wild-type

• Mechanistic implications:

  • Anisotropic B-factors indicate that the C4 atom of nicotinamide exhibits directional mobility in FNR:NADP+ complexes

  • This mobility aligns with predicted boat-like excursions of the nicotinamide ring that enhance hydride transfer efficiency

  • Active site compression is evidenced by packing interactions that favor catalytically productive conformations

To study similar effects in E. faecalis FNR:

• Generate equivalent tyrosine mutants through site-directed mutagenesis

• Perform pre-steady-state kinetic analyses using stopped-flow spectroscopy

• Determine hydride transfer rates using deuterated substrates to measure kinetic isotope effects

• Collect high-resolution crystal structures of the variants with bound substrates

• Analyze molecular dynamics simulations to quantify nicotinamide ring mobility

What transcriptional changes occur in the EF_2899 gene under infection-relevant growth conditions?

Under infection-relevant growth conditions, the transcriptional regulation of EF_2899 in Enterococcus faecalis exhibits complex patterns that can be studied through comprehensive transcriptomic approaches:

• Infection-relevant conditions to test:

  • Urinary tract infection model: Artificial urine medium with varying pH and osmolarity

  • Blood infection model: Serum or blood culture conditions

  • Biofilm formation: Static growth on appropriate surfaces

  • Antibiotic stress: Sub-inhibitory concentrations of clinically relevant antibiotics

  • Oxidative/nitrosative stress: H₂O₂ or NO· generating compounds

  • Nutrient limitation: Carbon, nitrogen, or phosphate restriction

• Methods for transcriptional analysis:

  • RNA sequencing (RNA-Seq) for global transcriptome analysis

  • Quantitative RT-PCR for targeted gene expression measurement

  • Promoter-reporter fusions (e.g., lacZ, gfp) for in vivo expression monitoring

  • CRISPR interference for functional validation

• Data analysis approaches:

  • Differential expression analysis between conditions

  • Time-course expression profiling

  • Co-expression network analysis to identify functional relationships

  • Integration with proteomics and metabolomics data

How does the crystal structure of EF_2899 inform potential inhibitor design for antimicrobial development?

The crystal structure of EF_2899 provides crucial insights for structure-based inhibitor design:

• Key structural features to target:

  • The FAD-binding pocket presents unique structural elements compared to human homologs

  • The nicotinamide-binding site contains bacterial-specific residues

  • The protein-protein interaction interface with ferredoxin offers selectivity

  • Active site compression regions identified in crystal structures reveal potential allosteric sites

• Rational inhibitor design approach:

  • Virtual screening against the NADP(H)-binding site

  • Fragment-based screening focusing on the active site

  • Structure-based design of transition state analogs that mimic the hydride transfer state

  • Development of covalent inhibitors targeting conserved cysteine residues

• Structural validation methods:

  • X-ray crystallography of enzyme-inhibitor complexes (target resolution <2.0 Å)

  • Hydrogen-deuterium exchange mass spectrometry to map binding interfaces

  • NMR studies to characterize binding dynamics

  • Molecular dynamics simulations to predict binding modes and conformational changes

Based on the available structural data from related FNRs, inhibitors that exploit the directionality of nicotinamide C4 atom mobility or that stabilize non-productive conformations of the enzyme would be particularly promising. The compressional distortion of FAD observed in crystal structures also suggests potential for developing compounds that disrupt this catalytically important feature .

What statistical approaches are appropriate for analyzing kinetic data from EF_2899 enzymological studies?

When analyzing kinetic data from EF_2899 enzymological studies, researchers should employ a comprehensive statistical framework:

• Preliminary data assessment:

  • Test for normality using Shapiro-Wilk or Kolmogorov-Smirnov tests

  • Identify and manage outliers using Grubbs' test or Dixon's Q-test

  • Assess homoscedasticity across substrate concentration ranges

• Kinetic parameter estimation:

  • Use nonlinear regression rather than linearized plots (avoid Lineweaver-Burk)

  • Apply weighted least squares methods when variance is heterogeneous

  • Determine confidence intervals for all kinetic parameters (Km, kcat, kcat/Km)

  • Consider bootstrap or jackknife resampling for robust parameter estimation

• Model selection and validation:

  • Compare different kinetic models using AIC (Akaike Information Criterion) or BIC (Bayesian Information Criterion)

  • Perform residual analysis to assess systematic deviations

  • Validate with independent datasets or cross-validation approaches

• Advanced statistical considerations:

  • Account for enzyme concentration uncertainty in kcat calculations

  • Consider global fitting for complex mechanistic models

  • Apply Bayesian methods for integrating prior knowledge

A recommended approach is to present kinetic data in both tabular and graphical formats. Tables should include best-fit parameters with confidence intervals, while graphs should display both experimental data points and fitted curves with residual plots .

How should researchers present and interpret spectroscopic data from EF_2899 studies?

Spectroscopic data from EF_2899 studies should be presented and interpreted using these guidelines:

• UV-Visible spectroscopy:

  • Present baseline-corrected spectra with clearly labeled axes and units

  • Include critical spectral features: FAD peaks (~450 nm, ~370 nm, ~270 nm)

  • When showing spectral changes, use difference spectra or 3D plots for time-course data

  • Quantify FAD content using extinction coefficients (ε450 ≈ 11,300 M⁻¹cm⁻¹)

  • Calculate A275/A450 ratio to assess protein purity (typically 5-6 for pure FNR)

• Fluorescence spectroscopy:

  • Report excitation/emission wavelengths, slit widths, and photomultiplier settings

  • Present normalized spectra when comparing different conditions

  • Use synchronous scanning for enhanced resolution of overlapping signals

  • Apply appropriate corrections for inner filter effects

  • Quantify quenching using Stern-Volmer analysis with statistical assessment

• Circular dichroism:

  • Express data in mean residue ellipticity units

  • Perform deconvolution analysis with multiple algorithms (SELCON, CONTINLL)

  • Report goodness-of-fit parameters for secondary structure estimates

  • Include thermal denaturation curves with calculated Tm values

  • Compare with predicted secondary structure from homology models

• Data presentation principles:

  • Use color schemes that are distinguishable in grayscale and for color-blind readers

  • Include error bars representing standard deviation or SEM

  • Annotate spectra with assignments for major features

  • Provide explanatory captions that can stand alone

For complex spectral changes during catalysis, consider using multivariate statistical methods such as principal component analysis (PCA) or singular value decomposition (SVD) to extract reaction components and kinetic phases .

How does E. faecalis Ferredoxin--NADP Reductase compare functionally to NADPH oxidases (NOXs)?

E. faecalis Ferredoxin--NADP Reductase (EF_2899) and NADPH oxidases (NOXs) share several functional similarities but differ in key aspects:

Despite these differences, both enzyme families catalyze FAD-enabled electron transfers involving NAD(P)H, making FNR a valuable prototype for understanding aspects of NOX function. The structural insights from FNR studies can be extrapolated to provide mechanistic information about the membrane-bound NOX enzymes, for which limited structural data is available .

When designing experiments to compare these enzymes:

• Use similar spectroscopic approaches to study FAD reduction/oxidation

• Apply stopped-flow techniques to resolve rapid electron transfer steps

• Consider the different physiological contexts when interpreting results

• Examine conserved active site residues across both enzyme families

What are the key differences in experimental approaches when studying EF_2899 compared to photosynthetic FNRs?

When studying E. faecalis FNR (EF_2899) versus photosynthetic FNRs, researchers should adapt their experimental approaches to account for several key differences:

• Physiological context:

  • Photosynthetic FNRs: Function in chloroplasts, primarily in the light-dependent reactions

  • EF_2899: Functions in bacterial metabolism, potentially in both aerobic and anaerobic conditions

• Electron donor specificity:

  • Photosynthetic FNRs: Highly specific for plant-type ferredoxins

  • EF_2899: May interact with bacterial ferredoxins with different reduction potentials

• Assay conditions:

  • Photosynthetic FNRs: Often studied under conditions mimicking chloroplast stroma (pH 7.5-8.0, higher Mg²⁺)

  • EF_2899: Should be studied under conditions relevant to bacterial cytoplasm (pH 7.0-7.5)

• Kinetic behaviors:

  • Photosynthetic FNRs: Often show higher catalytic efficiencies (kcat/Km)

  • EF_2899: May exhibit different substrate affinities and catalytic rates

• Redox potential considerations:

  • Photosynthetic FNRs: Tuned to function in photosynthetic electron transport chain

  • EF_2899: Adapted to bacterial redox environment and metabolic needs

• Experimental design adaptations:

  • Use appropriate bacterial ferredoxins as substrates instead of plant ferredoxins

  • Consider testing activity under both aerobic and anaerobic conditions

  • Examine potential roles in both NADPH production and consumption

  • Include physiologically relevant metabolites as potential regulators

  • Test pH optima across a broader range (pH 5.5-8.0)

These differences necessitate careful adaptation of experimental protocols when transferring methods developed for plant FNRs to the study of bacterial EF_2899 .

What are common challenges in obtaining active recombinant EF_2899 and how can they be overcome?

Researchers frequently encounter several challenges when producing active recombinant EF_2899:

• Incomplete FAD incorporation:

  • Symptoms: Low A450/A280 ratio, reduced specific activity

  • Solutions:

    • Add 10-100 μM FAD to expression media

    • Include FAD during protein purification

    • Perform reconstitution with excess FAD followed by dialysis

    • Optimize conditions for apoprotein refolding in the presence of FAD

• Protein aggregation:

  • Symptoms: Precipitation during purification, elution in void volume during size exclusion

  • Solutions:

    • Lower expression temperature (16-20°C)

    • Add stabilizing agents (5-10% glycerol, 0.5-1 M arginine during refolding)

    • Screen buffer conditions using differential scanning fluorimetry

    • Consider fusion partners (MBP, SUMO) to enhance solubility

• Proteolytic degradation:

  • Symptoms: Multiple bands on SDS-PAGE, decreasing yield during purification

  • Solutions:

    • Include protease inhibitors throughout purification

    • Add EDTA to chelate metal-dependent proteases

    • Minimize processing time and keep samples cold

    • Consider engineering construct to remove protease-sensitive regions

• Low enzymatic activity:

  • Symptoms: Purified protein shows poor catalytic performance

  • Solutions:

    • Verify protein folding using circular dichroism

    • Test activity under various buffer conditions (pH 6.0-8.5)

    • Ensure reducing environment with DTT or β-mercaptoethanol

    • Examine interaction with different ferredoxin partners

• Heterogeneous oxidation states:

  • Symptoms: Variable spectroscopic properties, inconsistent activity

  • Solutions:

    • Pre-oxidize or pre-reduce protein preparations

    • Perform anaerobic purification when necessary

    • Include appropriate redox buffers during storage

    • Characterize and account for different oxidation states in analyses

By systematically addressing these challenges, researchers can significantly improve the yield and quality of active recombinant EF_2899 for subsequent structural and functional studies .

How can researchers address data inconsistencies in EF_2899 kinetic measurements?

When confronted with data inconsistencies in EF_2899 kinetic measurements, researchers should implement a systematic troubleshooting approach:

• Identify the nature of inconsistencies:

  • Variation between technical replicates: Indicates procedural errors

  • Variation between biological replicates: Suggests sample preparation issues

  • Systematic deviations from expected models: May indicate mechanistic complexity

  • Time-dependent changes in activity: Points to enzyme stability problems

• Experimental validation steps:

  • Verify enzyme concentration using multiple methods (Bradford, BCA, A280)

  • Confirm FAD content spectroscopically and adjust for incomplete incorporation

  • Assess enzyme homogeneity by native PAGE or analytical size exclusion

  • Check for inhibitory contaminants in substrate preparations

  • Evaluate instrument calibration and detection linearity

• Statistical approaches to reconcile data:

  • Apply weighted regression methods if error magnitude varies with substrate concentration

  • Consider robust regression techniques less sensitive to outliers

  • Use bootstrapping to generate confidence intervals

  • Perform sensitivity analysis to identify influential data points

• Advanced troubleshooting measures:

  • Investigate potential substrate depletion or product inhibition

  • Test for hysteretic behavior using different pre-incubation conditions

  • Examine the influence of buffer components and ionic strength

  • Consider allosteric effects by testing for cooperativity

  • Evaluate the possibility of multiple enzyme forms or oxidation states

• Data reconciliation strategies:

  • Global fitting of multiple datasets with shared parameters

  • Hierarchical Bayesian modeling to account for batch-to-batch variation

  • Meta-analysis approaches when combining historical data

  • Transparent reporting of all data processing steps and exclusion criteria

By methodically addressing these aspects, researchers can resolve inconsistencies and develop more robust kinetic models for EF_2899 enzymatic activity .

What are promising research directions for understanding the physiological role of EF_2899 in E. faecalis virulence?

Several promising research directions could elucidate the physiological role of EF_2899 in E. faecalis virulence:

• Transcriptional profiling under infection conditions:

  • Conduct RNA-Seq analysis of E. faecalis under conditions mimicking different infection sites

  • Perform chromatin immunoprecipitation sequencing (ChIP-Seq) to identify regulators of EF_2899 expression

  • Use single-cell transcriptomics to assess population heterogeneity in EF_2899 expression during infection

• Genetic manipulation approaches:

  • Generate EF_2899 knockout and conditional mutants

  • Create reporter strains with EF_2899 promoter fusions to monitor expression in vivo

  • Utilize CRISPR interference for temporal control of EF_2899 expression

  • Develop complementation strains with site-directed mutants affecting catalytic activity

• Infection model studies:

  • Evaluate virulence of EF_2899 mutants in animal models of UTI, bacteremia, and endocarditis

  • Assess in vivo competitive fitness between wild-type and EF_2899 mutants

  • Examine biofilm formation capacity and antibiotic tolerance

  • Investigate host-pathogen interactions focusing on redox balance

• Metabolomic analyses:

  • Profile NADPH/NADP+ ratios in wild-type versus EF_2899 mutants

  • Conduct flux analysis to determine the contribution of EF_2899 to cellular NADPH pools

  • Examine metabolic adaptations in EF_2899 mutants under oxidative stress

  • Investigate potential role in supporting antioxidant defense systems

• Structural biology approaches:

  • Determine high-resolution structures of EF_2899 with physiological partners

  • Identify potential regulatory protein interactions through pull-down studies

  • Examine structural changes under different redox conditions

  • Investigate potential allosteric regulation by metabolites

These research directions would collectively provide a comprehensive understanding of EF_2899's role in E. faecalis physiology and pathogenesis, potentially revealing new therapeutic targets .

How might high-throughput screening approaches be designed to identify inhibitors of EF_2899?

An effective high-throughput screening (HTS) campaign to identify inhibitors of EF_2899 would involve:

• Assay development and optimization:

  • Primary enzymatic assay: Monitor NADPH formation/consumption spectrophotometrically at 340 nm

  • Alternative detection methods:

    • Fluorescence-based assays monitoring NADPH fluorescence (excitation 340 nm, emission 460 nm)

    • Coupled enzyme assays utilizing NADPH-dependent enzymes and colorimetric detection

    • Time-resolved fluorescence resonance energy transfer (TR-FRET) for binding assays

  • Assay validation parameters:

    • Z' factor >0.7 for robust screening

    • Signal-to-background ratio >5:1

    • Coefficient of variation <10%

    • DMSO tolerance up to 2%

• Compound library selection:

  • Diversity-oriented synthetic libraries (20,000-100,000 compounds)

  • Natural product extracts, particularly from antimicrobial sources

  • Fragment libraries for initial binding screens

  • Focus libraries targeting FAD or NADP(H)-binding enzymes

  • In silico pre-filtered libraries based on structural insights

• Screening cascade design:

  • Primary screen: Single-concentration inhibition assay (10-20 μM)

  • Confirmation screen: Dose-response curves for hits (IC50 determination)

  • Counter-screens:

    • Test against human FNR homologs to assess selectivity

    • Evaluate for compound interference with detection system

    • Assess aggregation potential using detergent-based controls

  • Mechanism characterization:

    • Determine inhibition modality (competitive, non-competitive, uncompetitive)

    • Measure binding kinetics using surface plasmon resonance

    • Evaluate effects on protein thermal stability

• Advanced hit characterization:

  • Structural studies of enzyme-inhibitor complexes

  • Cellular activity in E. faecalis growth/survival assays

  • Structure-activity relationship development

  • Assessment of bacterial specificity spectrum

  • Preliminary ADME and toxicity profiling

This systematic approach would efficiently identify, validate, and characterize potential inhibitors of EF_2899 with therapeutic potential as novel antimicrobial agents .