Recombinant Neurospora crassa Enolase (NCU10042)

What is the biochemical function of Neurospora crassa enolase?

Neurospora crassa enolase (NCU10042) functions as a metalloenzyme in the glycolytic pathway, catalyzing the conversion of 2-phosphoglycerate (2-PGA) to phosphoenolpyruvate (PEP). The standard enzymatic activity can be directly monitored by measuring the increase in PEP absorbance at 240 nm using spectrophotometry. One unit of enolase activity is defined as the amount of protein which catalyzes the formation of 1μmole PEP from 2-PGA in 1 minute under standard conditions (50 mM Tris pH 8.0, 0.1 M KCl, 0.5 mM 2-phosphoglycerate, and 1 mM MgSO4 at 25°C) . The reaction requires magnesium as a cofactor, which is crucial for stabilizing dimer contacts in the enzyme. Like other enolases, the N. crassa variant may also play additional roles beyond glycolysis, potentially functioning in cell wall formation and other cellular processes .

How do I express and purify recombinant N. crassa enolase?

Expression Protocol:

• Clone the N. crassa enolase gene (NCU10042) into an appropriate expression vector (e.g., pET-28a(+)) with an N-terminal His6 tag

• Transform into a suitable E. coli strain (e.g., BL21(DE3))

• Grow transformed cells in LB media with appropriate antibiotic at 37°C until reaching mid-log phase

• Induce protein expression with IPTG (typically 0.5-1.0 mM)

• Continue incubation at reduced temperature (16-25°C) for 12-16 hours

Purification Protocol:

• Harvest cells by centrifugation and resuspend in lysis buffer (50 mM Tris pH 8.0, 0.4 M NaCl, 5 mM β-mercaptoethanol, 200 μg/mL lysozyme, and protease inhibitors)

• Disrupt cells by sonication and clear lysate by centrifugation

• Apply filtered supernatant to a nickel affinity column equilibrated with buffer A (50 mM Tris pH 8.0, 0.4 M NaCl, 5 mM imidazole, 5 mM MgCl2)

• Wash column and elute with an imidazole gradient

• If desired, cleave the His-tag using an appropriate protease

• Perform size exclusion chromatography for final purification

What are the optimal buffer conditions for N. crassa enolase activity assays?

Standard Assay Conditions:

The activity of recombinant tagged and untagged enolase can be determined by direct monitoring of the increase in PEP absorbance at 240 nm. For calculating PEP concentration, use a molar extinction coefficient (Ɛ240 nm) of 1,300 M-1 cm-1 .

How does the structure of N. crassa enolase compare with enolases from other organisms?

While the specific crystal structure of N. crassa enolase is not detailed in the provided search results, comparative analysis with other fungal and bacterial enolases suggests several conserved structural features:

Expected Structural Features:

• Metal-binding sites (primarily for Mg2+)

• Substrate-binding pocket specific for 2-phosphoglycerate

• Dimer formation interface

• Catalytic residues conserved across species

For definitive structural analysis, crystallization could be performed using methods similar to those described for bacterial enolase:

• Concentrate protein to 10-12 mg/mL in buffer containing 20 mM HEPES pH 7.0, 50 mM NaCl, and 2 mM MgCl2

• Set up crystallization trials using hanging drop vapor diffusion

• Optimize conditions (a starting point could be 2.0 M ammonium sulfate, 0.1 M MES at pH 6.0, and 0.1M Na/K tartrate at 20°C)

• Both co-crystallization and soaking methods can be used to obtain substrate-bound structures

Thermal stability analysis using differential scanning calorimetry (DSC) could provide additional structural insights, with protocols involving heating purified protein from 0°C to 120°C at 1°C/min .

What genetic approaches can be used to study enolase function in N. crassa?

Gene Knockout/Modification Approaches:

• CRISPR-Cas9 System: While implementing CRISPR in N. crassa has been challenging due to its recalcitrance to expressing heterologous sequences, recent advances may overcome these limitations. The identification of heterologous expression positive (hep) mutants may facilitate CRISPR applications .

• Homologous Recombination: Traditional gene replacement using homologous recombination remains effective:

  • Construct gene replacement cassettes with appropriate selectable markers

  • Transform into N. crassa using electroporation

  • Screen transformants on selective media

  • Verify knockout by PCR and Southern blot analysis

  • Cross with wild-type to eliminate background mutations

• Sib Selection Procedure: For complementation of mutants:

  • Utilize high-efficiency transformation procedures (10,000-50,000 stable transformants per microgram of DNA)

  • Subdivide genomic libraries and test for complementation

  • Confirm gene identity using restriction site polymorphisms as genetic markers

• Endogenous Promoter Swap: For controlling expression levels while maintaining native regulation patterns.

How does enolase activity intersect with cell wall integrity pathways in N. crassa?

N. crassa enolase likely plays significant roles in cell wall integrity and remodeling based on functional studies in related systems. The Cell Wall Integrity (CWI) pathway in N. crassa involves:

• MAPK Cascade Components:

  • MIK-1 (NCU02234)

  • MEK-1 (NCU06419)

  • MAK-1 (NCU09842)

• Transcription Factors:

  • ADV-1 (NCU07392) is essential for cell wall stress response and directly activates transcription of elements involved in cell wall integrity

• Cell Wall Remodeling Enzymes:
  Research on the GUL-1 protein, which binds multiple RNAs involved in cell wall remodeling, identified several cell wall-related transcripts that likely intersect with enolase function:

  • 16 cell wall proteins

  • 15 glucanosyltransferases

  • α-1,3-glucan synthase (ags-1, NCU08132)

  • 5 gel transcripts (NCU08909, NCU07253, NCU01162, NCU06850, NCU06781)

  • Chitin synthase (chs-1, NCU03611)

Enolase may contribute to cell wall integrity through direct enzymatic activity or by affecting the expression or localization of these components. Additionally, enolase could influence CWI pathway activation, similar to how SSD1 mutations in S. cerevisiae result in constitutive CWI pathway activation .

What are the specific parameters for designing inhibitor studies with N. crassa enolase?

Inhibitor Screening Protocol:

• Sample Preparation:

  • Maintain constant enolase concentration (40 nM recommended)

  • Prepare inhibitors in 50 mM Tris pH 8.0 with 5% DMSO

  • Preincubate protein with inhibitor for 5 minutes in assay buffer (50 mM Tris pH 8.0, 0.1 M KCl, 1 mM MgSO4)

  • Initiate reaction with 2-PGA (1.0 mM final concentration)

• Measurement Parameters:

  • Monitor decrease in enolase activity at 15-second intervals for 4 minutes at 240 nm

  • Calculate IC50 values from initial rates of absorbance increase

  • Use appropriate positive control (Baker's yeast enolase recommended)

• Thermal Shift Assays:

  • Buffer exchange protein into 20 mM HEPES (pH 7.0), 50 mM NaCl, 2 mM MgCl2

  • Adjust protein and ligand concentrations to 6 μM and 60 μM respectively

  • Perform differential scanning calorimetry from 0°C to 120°C at 1°C/min

  • Analyze data using two-state transition model to determine Tm and ΔH values

Known enolase inhibitors that could be tested include ENOblock, which has shown efficacy in inhibiting enolase activity in other systems and has demonstrated neuroprotective effects .

How can recombinant N. crassa enolase be used to study broader fungal biology questions?

Recombinant N. crassa enolase serves as an excellent model for investigating fundamental aspects of fungal biology:

• Comparative Glycolytic Metabolism:

  • Analyze differences in kinetic parameters between fungal enolases to understand metabolic adaptations

  • Investigate the role of enolase in carbon source utilization and metabolic flexibility

• Morphogenesis and Development:
  N. crassa has complex developmental programs including hyphal elongation, branching, and conidiation. Enolase may play roles in these processes through:

  • Energy metabolism for rapid hyphal extension

  • Potential moonlighting functions in cell wall synthesis

  • Connections to signaling pathways that regulate development

• Stress Response Mechanisms:

  • Investigate enolase regulation during various stresses (e.g., glucose starvation, oxidative stress)

  • Examine connections to circadian rhythms and light responses, which are well-characterized in N. crassa

• Protein Targeting and Modification:

  • Study post-translational modifications of enolase

  • Investigate potential localization changes during different growth conditions or developmental stages

• Fungal-Specific Drug Target Identification:

  • Compare with human enolases to identify unique structural features

  • Develop selective inhibitors against fungal enolases with potential antifungal applications

What is the role of enolase in N. crassa endocytosis and membrane trafficking?

The potential role of enolase in N. crassa endocytosis and membrane trafficking represents an interesting area for investigation. While direct evidence is limited in the provided search results, several connections can be made:

• Endocytic Machinery in N. crassa:
  N. crassa possesses a complex endocytic system involving:

  • Actin-dependent endocytosis mechanisms

  • Clathrin-mediated endocytosis

  • Key endocytic proteins including WASP, clathrin light chain, and Rab5

• Potential Interactions with Trafficking Machinery:
  Enolase may interact with:

  • Components of early endosomes

  • Vesicle transport machinery

  • Membrane-associated proteins

• Experimental Approaches:

  • Fluorescently tag enolase to visualize localization during endocytosis

  • Use FM4-64 as an endocytic marker to track potential co-localization

  • Apply inhibitors like Brefeldin A (which blocks vesicular transport to the Spitzenkörper) to assess effects on enolase localization

• Membrane Association:
  In other systems, enolase has been found on cell surfaces and in association with membrane structures. Specific experimental protocols could include:

  • Cell fractionation to determine membrane association

  • Immunofluorescence to detect external/surface-exposed enolase

  • Co-immunoprecipitation with known membrane trafficking components

How does enolase contribute to the circadian regulatory system in N. crassa?

N. crassa is a model organism for studying circadian rhythms, and metabolic enzymes like enolase may play significant roles in this system:

• Circadian Clock Components in N. crassa:
  The core components include:

  • White-Collar Complex (WCC) composed of WC-1 and WC-2 (GATA-type transcription factors)

  • Frequency (FRQ)

  • FRQ-FRH-CK1a complex (the negative feedback element)

• Metabolic Connections to Circadian Rhythms:

  • GUL-1 protein binds at least 17 circadian clock-associated transcripts

  • Glucose levels affect many signal transduction pathways and may challenge the TTFL-based circadian clock

  • Metabolic enzymes like enolase could provide feedback on cellular energy status

• Research Approaches:

  • Examine enolase expression patterns over circadian time

  • Analyze the effects of altered enolase activity on clock component function

  • Investigate potential protein-protein interactions between enolase and clock components

  • Study the impacts of carbon source availability on these interactions

• Nutrient Compensation Mechanisms:
  Molecular mechanisms of nutrient compensation have been intensively studied in N. crassa , and enolase may contribute to maintaining clock function during nutrient fluctuations through its role in carbon metabolism.

What are the optimal conditions for crystallizing N. crassa enolase for structural studies?

While the search results don't provide specific crystallization conditions for N. crassa enolase, the following protocol can be adapted from successful crystallization of bacterial enolase:

Recommended Crystallization Protocol:

• Protein Preparation:

  • Purify recombinant enolase to >95% homogeneity

  • Buffer exchange into 20 mM HEPES pH 7.0, 50 mM NaCl, and 2 mM MgCl2

  • Concentrate to 10-12 mg/mL

• Initial Screening:

  • Set up crystallization trials using commercial sparse matrix screens

  • Utilize hanging drop vapor diffusion method with 1:1 ratio of protein to reservoir solution

  • Incubate at 20°C

• Optimization Based on Bacterial Enolase Conditions:

  Parameter	Starting Condition
  Protein concentration	12 mg/mL
  Buffer	20 mM HEPES pH 7.0
  Salt	50 mM NaCl
  Precipitant	2.0 M ammonium sulfate
  Additives	0.1 M MES pH 6.0, 0.1 M Na/K tartrate
  Temperature	20°C
  Method	Hanging drop

• Substrate/Ligand Co-crystallization:

  • For substrate-bound structures, incubate protein with 2.5 mM 2-PGA for 2 hours on ice prior to setting up trays

  • Alternatively, soak preformed crystals in mother liquor containing 2.5 mM substrate overnight

• Data Collection Considerations:

  • Cryoprotect crystals using mother liquor supplemented with 20-25% glycerol or ethylene glycol

  • Flash-freeze in liquid nitrogen

  • Collect diffraction data at 100K using synchrotron radiation

How can I determine if my recombinant N. crassa enolase is properly folded and active?

Comprehensive Activity and Folding Assessment:

• Enzymatic Activity Assay:

  • Measure conversion of 2-PGA to PEP spectrophotometrically at 240 nm

  • Calculate specific activity (units per mg protein)

  • Compare to published values for other fungal enolases

  • Determine Km and Vmax parameters

• Thermal Stability Analysis:

  • Perform differential scanning calorimetry (DSC)

  • Heat protein sample from 0°C to 120°C at 1°C/min

  • Determine melting temperature (Tm) and enthalpy change (ΔH)

  • Compare with and without substrate or cofactors

• Structural Characterization:

  • Circular dichroism (CD) to assess secondary structure content

  • Size exclusion chromatography to verify oligomeric state (expected dimer)

  • Dynamic light scattering to check for aggregation

• Metal Binding Analysis:

  • Inductively coupled plasma mass spectrometry (ICP-MS) to quantify bound metals

  • Activity assays with and without EDTA to assess metal dependence

  • Reconstitution experiments with various divalent metals

• Functional Response to Known Inhibitors:

  • Test inhibition with known enolase inhibitors

  • Calculate IC50 values

  • Compare inhibition profile with other characterized enolases

What are the key considerations for designing site-directed mutagenesis studies of N. crassa enolase?

Comprehensive Site-Directed Mutagenesis Strategy:

• Target Selection Rationale:

  • Catalytic residues based on structural homology with other enolases

  • Metal-binding sites critical for activity

  • Substrate recognition residues

  • Dimer interface residues

  • Surface residues potentially involved in protein-protein interactions

  • Conserved residues identified through multiple sequence alignments

• Mutation Design Principles:

  • Conservative substitutions to probe specific interactions

  • Charge reversal mutations to disrupt electrostatic interactions

  • Alanine scanning to identify essential side chains

  • Introduction of bulky residues to create steric hindrance

  • Cysteine mutations for subsequent chemical modification

• Experimental Validation Workflow:

  • Expression and purification of mutant proteins

  • Activity assays comparing wild-type and mutant enzymes

  • Thermal stability analysis via DSC

  • Structural characterization (CD, size exclusion chromatography)

  • Binding studies with substrates, cofactors, and inhibitors

• Advanced Characterization:

  • Pre-steady state kinetics to identify rate-limiting steps

  • pH-rate profiles to probe ionization states

  • Solvent isotope effects to investigate proton transfer events

  • Temperature dependence studies to calculate activation parameters

• In vivo Complementation:

  • Test ability of mutants to rescue phenotypes in enolase-deficient strains

  • Analyze effects on growth, development, and stress responses

How can I investigate potential moonlighting functions of N. crassa enolase beyond its glycolytic role?

Comprehensive Strategy to Identify Moonlighting Functions:

• Subcellular Localization Analysis:

  • Create GFP or other fluorescent protein fusions

  • Perform immunofluorescence with anti-enolase antibodies

  • Conduct subcellular fractionation followed by western blotting

  • Look for non-cytosolic localization (membrane, nucleus, cell wall)

• Protein-Protein Interaction Studies:

  • Co-immunoprecipitation with candidate interacting proteins

  • Yeast two-hybrid screening

  • Proximity labeling approaches (BioID or APEX)

  • Mass spectrometry analysis of protein complexes

  • Investigate potential interactions with cell wall remodeling enzymes, signaling complexes, or cellular transport machinery

• Surface Exposure Analysis:

  • Cell surface biotinylation followed by pulldown and western blotting

  • Flow cytometry with anti-enolase antibodies on intact cells

  • Immunoelectron microscopy

  • Protease shaving of intact cells followed by mass spectrometry

• Functional Studies:

  • RNA-binding assays (similar to GUL-1 protein studies in N. crassa)

  • DNA-binding capability assessment

  • Plasminogen binding analysis (demonstrated for enolases in other systems)

  • Evaluation of effects on cell wall integrity and structure

• Phenotypic Analysis Under Various Conditions:

  • Stress responses (oxidative, heat, cell wall stress)

  • Developmental transitions

  • Nutrient limitation

  • Circadian time points

What are the significant differences between N. crassa enolase and other fungal enolases that should inform my experimental design?

While the search results don't provide comprehensive comparative information specifically about N. crassa enolase versus other fungal enolases, several important considerations can be inferred:

• Genomic Context and Regulation:

  • N. crassa has complex genome defense mechanisms including repeat-induced point mutation (RIP), quelling, and meiotic silencing that may affect heterologous expression

  • Position of the enolase gene (NCU10042) in the genome context may influence its regulation

  • Potential involvement in circadian regulation may differ from yeasts like S. cerevisiae

• Structural Considerations:

  • N. crassa is a filamentous fungus with different cellular organization compared to yeasts

  • Potential unique structural features adapted to filamentous growth

  • Possible differences in metal binding preferences or cofactor requirements

• Functional Adaptations:

  • N. crassa undergoes complex development including conidiation and sexual cycles

  • The role of enolase may extend to specialized structures such as the Spitzenkörper in hyphal tips

  • Potential involvement in cell wall dynamics specific to filamentous fungi

• Experimental Design Implications:

  Parameter	Consideration
  Expression systems	Consider N. crassa expression systems for proper folding and modification
  Buffer conditions	May require optimization specific to N. crassa enolase
  Temperature stability	Assess thermal profiles relevant to N. crassa growth conditions
  Protein-protein interactions	Investigate interactions with filamentous fungi-specific partners
  Cellular assays	Use N. crassa rather than yeast-based assays when possible

• Conservation Analysis:
  Perform detailed sequence and structural alignments with enolases from:

  • Other filamentous fungi (e.g., Aspergillus)

  • Yeasts (S. cerevisiae, S. pombe)

  • Bacteria and mammals for broader comparison

How can recombinant N. crassa enolase be used as a model to understand broader metabolic regulation in filamentous fungi?

Recombinant N. crassa enolase provides an excellent model system for investigating metabolic regulation in filamentous fungi:

• Carbon Metabolism Networks:

  • Use enolase activity as a readout for glycolytic flux under different carbon sources

  • Investigate regulatory mechanisms in response to glucose availability

  • Examine connections between glycolysis and other metabolic pathways

  • Study metabolic adaptation during different developmental stages

• Stress Response Integration:

  • Analyze enolase regulation during oxidative stress

  • Investigate connections to cell wall stress responses

  • Examine coordination with mitochondrial energy metabolism

  • Study the relationship between metabolic enzymes and programmed cell death pathways

• Developmental Stage-Specific Regulation:

  • Compare enolase activity during vegetative growth versus reproductive stages

  • Analyze potential post-translational modifications across developmental transitions

  • Investigate changes in protein-protein interactions during different life cycle stages

• Circadian and Light-Responsive Metabolism:

  • Use N. crassa's well-characterized circadian system to study temporal regulation of metabolism

  • Investigate how light signals influence metabolic enzyme activity

  • Examine connections between clock components and metabolic regulation

• Comparative Approaches:

  • Develop systems for parallel analysis of enolase from multiple fungal species

  • Identify conserved versus specialized regulatory mechanisms

  • Create chimeric enzymes to pinpoint regions responsible for filamentous fungi-specific properties

What role does enolase play in the adaptation of N. crassa to nutrient limitation conditions?

Enolase likely plays significant roles in the adaptation of N. crassa to nutrient limitation, though specific mechanisms must be investigated:

• Glucose Starvation Response:

  • N. crassa shows robust molecular timekeeping even under severe carbon limitation, suggesting adaptive metabolic regulation

  • Enolase activity may be modulated to maintain essential energy production while conserving resources

  • Investigation should examine enolase expression, post-translational modifications, and activity under glucose limitation

• Metabolic Reprogramming Mechanisms:

  • During nutrient stress, N. crassa may redirect carbon flux through alternative pathways

  • Enolase might participate in regulatory feedback loops sensing energy status

  • Potential moonlighting functions of enolase could become more prominent under stress conditions

• Gene Expression Changes:
  Research shows that treatment of N. crassa with phytosphingosine (PHS) induces changes in expression patterns of metabolic genes :

  • Increased expression of genes involved in glyoxylate cycle and lipid metabolism

  • Downregulation of mitochondrial protein genes

  • Similar patterns might occur during nutrient limitation

• Experimental Approaches:

  • Transcriptomic analysis comparing enolase expression across nutrient conditions

  • Metabolomic profiling to track carbon flux through glycolysis

  • Phenotypic analysis of strains with altered enolase expression under nutrient limitation

  • Protein-protein interaction studies under nutrient-rich versus limited conditions

• Connection to Development:

  • Nutrient limitation often triggers developmental transitions in fungi

  • Enolase activity changes may signal or respond to these transitions

  • Investigation should examine enolase during transitions to conidiation or sexual development triggered by nutrient stress

How can studies of N. crassa enolase inform our understanding of fungal pathogenicity mechanisms?

Although N. crassa is not a pathogen, studies of its enolase can provide valuable insights into fungal pathogenicity mechanisms:

• Surface Display and Host Interaction:

  • In pathogens, enolase is often displayed on the cell surface and binds host plasminogen

  • Comparative analysis of N. crassa enolase with pathogenic fungal enolases can identify structural features important for host interactions

  • Investigation of potential surface localization mechanisms in N. crassa may reveal conserved trafficking pathways

• Cell Wall Integrity and Remodeling:

  • Cell wall structure and integrity are critical virulence determinants

  • N. crassa studies show connections between metabolic enzymes and cell wall remodeling

  • The Cell Wall Integrity (CWI) pathway components in N. crassa have homologs in pathogenic fungi

  • Understanding how enolase intersects with these pathways provides insights into invasive growth mechanisms

• Metabolic Adaptation During Infection:

  • Pathogens must adapt to variable nutrient conditions during infection

  • N. crassa's adaptations to nutrient limitation can serve as models for similar processes in pathogens

  • Comparative studies of regulatory mechanisms may identify conserved strategies

• Protein Moonlighting in Virulence:

  • Non-glycolytic functions of enolase often contribute to pathogenicity

  • Identifying moonlighting functions in N. crassa enolase can guide searches for similar functions in pathogens

  • Structural features enabling moonlighting may be conserved across species

• Experimental Approaches:

  • Express pathogen enolases in N. crassa to study functional conservation

  • Create chimeric proteins to identify regions conferring specific properties

  • Develop screening systems in N. crassa to test inhibitors targeting pathogen enolases

How does post-translational modification affect the function and localization of N. crassa enolase?

The search results don't provide specific information about post-translational modifications (PTMs) of N. crassa enolase, but based on studies in other systems, several important PTMs likely regulate its function:

• Potential Modifications:

  • Phosphorylation: Likely regulates activity and protein-protein interactions

  • Acetylation: May affect catalytic activity and subcellular localization

  • Glycosylation: Could influence surface exposure and extracellular functions

  • Proteolytic processing: Specific cleavage might activate moonlighting functions (similar to cathepsin X cleavage of NSE C-terminal in other systems)

• Regulatory Significance:

  • PTMs may form a code that determines which cellular function enolase performs

  • Modifications could respond to metabolic or environmental changes

  • Different modifications might predominate during specific developmental stages

• Localization Effects:

  • Surface exposure may depend on specific PTMs

  • Nuclear, mitochondrial, or other non-cytosolic localization might be PTM-directed

  • Membrane association could be regulated through modifications

• Experimental Approach:

  • Mass spectrometry analysis to map comprehensive PTM profile

  • Site-directed mutagenesis of modified residues

  • Phosphoproteomics under different growth conditions

  • Comparison of PTM patterns during development and stress responses

  • Analysis of effects of specific PTMs on enzymatic activity and protein interactions

• PTM Crosstalk:

  • Investigate how different modifications influence each other

  • Study whether specific signaling pathways target enolase for modification

  • Examine connections between modifications and protein stability or turnover

What insights can systems biology approaches provide about the role of enolase in N. crassa metabolic networks?

Systems biology approaches can provide comprehensive insights into the role of enolase within the broader metabolic network of N. crassa:

• Multi-omics Integration:

  • Combine transcriptomics, proteomics, metabolomics, and fluxomics data

  • Map enolase connections within the metabolic network

  • Identify condition-specific regulatory patterns

  • Construct predictive models of glycolytic flux

• Network Analysis:

  • Position enolase in protein-protein interaction networks

  • Identify metabolic modules connected to enolase function

  • Explore regulatory networks controlling enolase expression

  • Map genetic interactions through systematic genetic perturbations

• Temporal and Developmental Dynamics:

  • Track changes in enolase activity across the N. crassa life cycle

  • Investigate circadian regulation of metabolic networks

  • Monitor adaptation to environmental changes

  • Study metabolic remodeling during development

• Comparative Systems Analysis:

  • Compare N. crassa metabolic network architecture with other fungi

  • Identify conserved and divergent features

  • Relate network differences to ecological and evolutionary adaptations

• Computational Modeling:

  • Develop constraint-based models (e.g., flux balance analysis)

  • Create kinetic models of glycolysis incorporating enolase parameters

  • Build genome-scale metabolic models

  • Simulate perturbations to predict systemic effects of enolase modulation

This systems-level understanding would position enolase within its broader functional context and reveal emergent properties not apparent from reductionist approaches.

What are the most reliable genetic resources for studying enolase in N. crassa?

Genetic Resources and Tools for N. crassa Enolase Research:

• Strain Resources:

  • Fungal Genetics Stock Center (FGSC): Maintains the most comprehensive collection of N. crassa strains

  • Neurospora Genome Project knockout collection: Includes strains with mutations in genes related to metabolism

  • N. crassa wild-type strains: Standard laboratory strains include 74-OR23-1A and 74-OR8-1a

• Genomic Resources:

  • Neurospora crassa genome database at the Broad Institute

  • FungiDB: Integrated genomic database for fungi including N. crassa

  • MycoCosm (JGI): Comparative genomics platform for fungi

• Genetic Manipulation Tools:

  • Gene replacement cassettes for homologous recombination

  • Sib selection procedure for cloning genes by complementation of mutants

  • Heterologous expression strategies accounting for N. crassa's genome defense mechanisms

  • Transformation protocols optimized for high efficiency (10,000-50,000 transformants per μg DNA)

• Expression Systems:

  • pMF272 vector series for GFP tagging

  • qa-2 regulatable promoter system

  • ccg-1 constitutive promoter constructs

  • his-3 targeting vectors for stable integration

• Relevant Methodologies:

  • Protocols for electroporation transformation of N. crassa

  • Methods for genomic DNA extraction from N. crassa mycelium

  • Protein extraction and western blotting protocols

  • Crossing protocols for genetic analysis

What are the current technical challenges in studying N. crassa enolase and how can they be addressed?

Major Technical Challenges and Solutions:

• Heterologous Expression Challenges:

  • Challenge: N. crassa's recalcitrance to expressing ectopic sequences

  • Solutions:

    • Use of hep (heterologous expression positive) mutants

    • Codon optimization for N. crassa expression

    • Integration of constructs at specific genomic loci

    • Use of strong endogenous promoters

• Protein Purification Issues:

  • Challenge: Potential instability or insolubility during purification

  • Solutions:

    • Optimize buffer conditions with appropriate cofactors (e.g., Mg2+)

    • Use affinity tags compatible with enzymatic activity

    • Employ mild detergents for membrane-associated fractions

    • Consider native purification approaches from N. crassa

• Functional Redundancy:

  • Challenge: Potential compensatory mechanisms masking phenotypes

  • Solutions:

    • Create multiple isozyme knockouts

    • Use conditional expression systems

    • Employ acute inhibition approaches

    • Perform biochemical analyses in addition to genetic studies

• Genome Defense Mechanisms:

  • Challenge: Silencing of introduced sequences through quelling or RIP

  • Solutions:

    • Strategic design of constructs to avoid repetitive sequences

    • Use of strains defective in silencing mechanisms

    • Careful monitoring of transgene expression over time

    • Integration at permissive genomic loci

• Advanced Structural Analysis:

  • Challenge: Obtaining high-resolution structural data

  • Solutions:

    • Optimize crystallization conditions specifically for N. crassa enolase

    • Consider cryo-EM for difficult-to-crystallize forms

    • Use molecular dynamics simulations to complement experimental data

    • Perform comparative modeling using bacterial enolase structures as templates