Recombinant Aspergillus clavatus NADH-cytochrome b5 reductase 2 (mcr1)

What is NADH-cytochrome b5 reductase 2 and what are its primary functions?

NADH-cytochrome b5 reductase 2 (MCR1) is a flavoenzyme that catalyzes the reduction of two molecules of cytochrome b5 using NADH as the physiological electron donor . The enzyme belongs to the oxidoreductase family and plays crucial roles in various metabolic pathways. In fungi like Aspergillus species, MCR1 is involved in mitochondrial electron transport and can function as a mitochondrial cytochrome b reductase .

The general reaction catalyzed by this enzyme is:
NADH + 2 cytochrome b5 (oxidized) → NAD+ + 2 cytochrome b5 (reduced)

The enzyme contains flavin as a prosthetic group that facilitates electron transfer from NADH to cytochrome b5. This reaction is essential for several cellular processes including fatty acid metabolism, steroid biosynthesis, and drug metabolism in various organisms .

What is the typical structure of fungal NADH-cytochrome b5 reductase 2?

Fungal NADH-cytochrome b5 reductase 2 proteins typically consist of 290-310 amino acid residues as seen in homologous proteins from various species . The protein has two main functional domains:

• A flavin-binding domain (FAD-binding domain): This contains a β-barrel structure that holds the FAD prosthetic group

• An NADH-binding domain: This is responsible for binding and oxidizing NADH

Structural analysis of cytochrome b5 reductases across species reveals a conserved arrangement of three key amino acid residues (arginine, tyrosine, and serine) in the flavin-binding domain that form hydrogen bonds with the flavin molecule . This structural feature is critical for the enzyme's function and is likely conserved in Aspergillus clavatus MCR1 as well.

The general domain organization of the protein includes:

• N-terminal membrane-binding region (in some forms)

• FAD-binding domain with characteristic β-barrel fold

• NADH-binding domain with Rossmann fold

How do I express and purify recombinant Aspergillus clavatus MCR1?

Expression and purification of recombinant Aspergillus clavatus MCR1 typically follows protocols similar to those used for other fungal cytochrome b5 reductases:

Expression System:

• E. coli is commonly used as an expression system for fungal proteins, including MCR1

• The coding sequence should be cloned into an appropriate expression vector with a His-tag or other affinity tag for purification

• Expression is typically induced in bacterial cultures grown to mid-log phase

Purification Protocol:

• Harvest cells and lyse using appropriate buffer (typically Tris-based buffer pH 8.0)

• Clarify lysate by centrifugation

• Perform affinity chromatography using Ni-NTA resin for His-tagged protein

• Elute with imidazole gradient

• Further purify using ion exchange and/or size exclusion chromatography if needed

• Verify purity by SDS-PAGE (should be >90% pure)

Storage Conditions:

• Store purified protein at -20°C/-80°C

• For long-term storage, add glycerol to a final concentration of 50%

• Avoid repeated freeze-thaw cycles

• Working aliquots can be stored at 4°C for up to one week

What are the optimal buffer conditions for MCR1 enzyme activity assays?

Optimal buffer conditions for MCR1 enzyme activity assays typically include:

Buffer Composition:

• Tris or phosphate buffer (50-100 mM) at pH 7.0-8.0

• NaCl (50-150 mM) for ionic strength

• Sometimes supplemented with glycerol (5-10%) for protein stability

Reaction Components:

• NADH (50-200 μM) as electron donor

• Cytochrome b5 (10-50 μM) as electron acceptor

• FAD (1-10 μM) may be added to ensure full enzyme activity

• Enzyme concentration typically 0.1-1.0 μg/ml

Measurement Conditions:

• Activity is usually measured spectrophotometrically by following:

  • NADH oxidation at 340 nm (decrease in absorbance)

  • Cytochrome b5 reduction at 424 nm (increase in absorbance)

• Temperature: 25-30°C

• Reactions initiated by addition of enzyme or substrate

Calculation of Activity:

• Activity calculated using extinction coefficients:

  • NADH: ε340 = 6,220 M−1 cm−1

  • Reduced cytochrome b5: Δε424 ≈ 100,000 M−1 cm−1

How do mutations in conserved residues affect the kinetic parameters of MCR1?

Mutations in conserved residues of MCR1 can significantly alter the enzyme's kinetic parameters. Based on structure-function studies of cytochrome b5 reductases, several key observations can be made:

Effects on NADH Binding:
Mutations of conserved lysine residues (equivalent to K83 in rat cb5r) that interact with NADH through electrostatic interactions can:

• Increase the apparent Km for NADH (reduced binding affinity)

• Decrease the apparent kcat (reduced catalytic efficiency)

• Have minimal effects on Km for cytochrome b5

For example, glutamate substitution at this position produces an enzyme that is less efficient at NADH utilization compared to wild type, suggesting this residue plays a crucial role in stabilizing the NADH-bound form of the enzyme .

Effects on Flavin Binding:
Mutations in the conserved arginine-tyrosine-serine triad that forms hydrogen bonds with the flavin can:

Mutation Effects on Kinetic Parameters:

These patterns are based on studies of related cytochrome b5 reductases and would be expected to apply to Aspergillus clavatus MCR1 given the conserved nature of the enzyme's structure and function .

How can I perform comparative structural analysis between MCR1 from different fungal species?

Comparative structural analysis of MCR1 from different fungal species involves several methodological approaches:

Sequence Alignment and Phylogenetic Analysis:

• Collect MCR1 protein sequences from target fungal species including Aspergillus clavatus, Mortierella alpina, and Saccharomyces cerevisiae

• Perform multiple sequence alignment using tools like MUSCLE, CLUSTAL, or T-COFFEE

• Identify conserved domains and critical residues (particularly the flavin-binding β-barrel domain)

• Generate phylogenetic trees to examine evolutionary relationships

• Analyze conservation specifically in the flavin-binding triad (arginine, tyrosine, and serine) which is crucial for function

Homology Modeling:

• Use resolved structures of cytochrome b5 reductases (e.g., rat cb5r at 2.0 Å resolution ) as templates

• Generate homology models using software like SWISS-MODEL, Phyre2, or MODELLER

• Evaluate model quality using PROCHECK, VERIFY3D, or MolProbity

• Compare barrel-folding patterns across different fungal MCR1 models

• Analyze specific arrangements of the three highly conserved amino acid residues that interact with flavin

Structural Comparison Metrics:

• RMSD (Root Mean Square Deviation) calculations for backbone atoms

• Analysis of electrostatic surface potentials

• Hydrogen bonding networks, particularly in the active site

• Solvent accessibility of key functional residues

• Location and orientation of NAD+/NADH binding sites

What are the key differences between mitochondrial and microsomal forms of cytochrome b5 reductase in fungi?

In fungi, including Aspergillus species, cytochrome b5 reductase can exist in different cellular compartments, primarily as mitochondrial and microsomal forms. Understanding these differences is important for research applications:

Structural Differences:

• Mitochondrial MCR1 typically contains an N-terminal signal sequence that targets it to the mitochondria

• Microsomal forms generally have a membrane-binding domain for association with the endoplasmic reticulum

• The core catalytic domains remain largely conserved between the two forms

Functional Differences:

• Mitochondrial MCR1 is involved in electron transport processes specific to mitochondria, functioning as a mitochondrial cytochrome b reductase

• Microsomal forms participate in fatty acid desaturation, cholesterol biosynthesis, and drug metabolism

• The two forms may have different electron accepting partners beyond cytochrome b5

Regulation and Expression:

• The two forms are often encoded by different genes or result from alternative splicing or translation initiation

• Expression patterns differ based on cellular conditions and metabolic demands

• Post-translational modifications may differ between mitochondrial and microsomal forms

Research Implications:

• When working with recombinant MCR1, researchers should verify which form they are expressing

• Functional assays should consider the natural electron acceptors of the specific form

• Subcellular localization studies should be conducted to confirm proper targeting in heterologous expression systems

How does Aspergillus clavatus MCR1 compare to other Aspergillus species in terms of sequence conservation and potential functional differences?

While specific data on Aspergillus clavatus MCR1 is limited in the search results, we can infer likely characteristics based on knowledge of related Aspergillus species and general patterns of conservation in fungal cytochrome b5 reductases:

Sequence Conservation Analysis:
Aspergillus species typically show high conservation in core functional domains of enzymes. Based on comparative genomics studies of other Aspergillus enzymes:

• Core catalytic domains (FAD and NADH binding regions) are typically >80% conserved across Aspergillus species

• N-terminal regions often show greater variability, which may relate to differences in subcellular targeting

• The flavin-binding triad (arginine, tyrosine, and serine) would be expected to be 100% conserved

Clade-Specific Variations:
Aspergillus species can be grouped into different clades that may show clade-specific variations:

• A. fumigatus and related species often cluster in phylogenetic analyses

• A. clavatus would likely group with other members of its clade

• These groupings may correlate with functional specializations of enzymes including MCR1

Potential Functional Differences:

• Substrate specificity may vary slightly between species

• Kinetic parameters (Km, kcat) may differ based on adaptation to different ecological niches

• Post-translational regulation mechanisms might vary between species

Research Recommendations:

• Perform detailed sequence alignments between Aspergillus species MCR1 proteins

• Consider strain variations within A. clavatus that might affect MCR1 function

• When making experimental comparisons, account for potential differences in optimal pH, temperature, and buffer conditions

What are the optimal expression conditions for high-yield production of recombinant Aspergillus clavatus MCR1?

Based on protocols for similar fungal proteins, the following conditions can be optimized for high-yield production of recombinant Aspergillus clavatus MCR1:

Expression System Optimization:

Co-expression Strategies:

• Co-expression with chaperones (GroEL/GroES, DnaK/DnaJ) may improve folding

• Co-expression with FAD synthase may improve flavin incorporation

• Supplementing growth medium with riboflavin (10-20 mg/L) can enhance FAD availability

Solubility Enhancement:

• Addition of 0.1-0.5% Triton X-100 to lysis buffer

• Including 5-10% glycerol in buffers

• Adding stabilizing agents like trehalose (6%)

• Maintaining pH in the 7.5-8.0 range

Yield Optimization:

• Typical yields of 5-20 mg per liter of culture are achievable

• Optimization may increase yields to >30 mg per liter

• Final product should have >90% purity as determined by SDS-PAGE

How can I investigate the role of MCR1 in Aspergillus clavatus drug resistance mechanisms?

Investigating the role of MCR1 in Aspergillus clavatus drug resistance mechanisms requires a multifaceted approach:

Gene Expression Analysis:

• Compare MCR1 expression levels between drug-resistant and susceptible strains using RT-qPCR

• Perform RNA-seq to identify co-regulated genes in resistance pathways

• Analyze MCR1 expression in response to antifungal challenges at different timepoints

Genetic Manipulation Approaches:

• Generate MCR1 knockout mutants using CRISPR-Cas9 or traditional homologous recombination

• Create MCR1 overexpression strains

• Develop point mutations in key residues based on structural insights

• Test antifungal susceptibility of these mutants compared to wild type

Functional Assays:

• Measure MCR1 enzyme activity in membrane fractions of resistant vs. susceptible strains

• Assess intracellular redox state changes in different strains

• Monitor electron transport efficiency and mitochondrial function

• Evaluate cellular responses to oxidative stress induced by antifungals

Comparative Genomics:

• Analyze MCR1 sequence variations in drug-resistant clinical isolates

• Look for SNPs or structural variations that correlate with resistance phenotypes

• Compare with patterns observed in other drug-resistant Aspergillus species, such as A. fumigatus

• Construct phylogenetic trees to identify lineage-specific adaptations

Methodological Considerations:

• Control for genetic background differences beyond MCR1 variations

• Use multiple drug classes to distinguish specific vs. general resistance mechanisms

• Include time-course studies to capture adaptive responses

• Validate findings across multiple independent strains

What are the most reliable methods for determining the kinetic parameters of MCR1?

Reliable determination of kinetic parameters for MCR1 requires careful experimental design and analysis:

Steady-State Kinetics:

• Use spectrophotometric assays monitoring:

  • NADH oxidation at 340 nm (ε = 6,220 M−1 cm−1)

  • Cytochrome b5 reduction at 424 nm (Δε ≈ 100,000 M−1 cm−1)

• Maintain temperatures between 25-30°C

• Use initial rate measurements (<10% substrate consumption)

• Vary one substrate concentration while keeping others saturating

• Fit data to appropriate enzyme kinetic models (Michaelis-Menten, ping-pong, etc.)

Rapid Kinetics Approaches:

• Stopped-flow spectroscopy to measure:

  • Flavin reduction rates

  • Cytochrome b5 reduction rates

  • Intermediate formation/decay

• Temperature-jump methods for conformational changes

• Quenched-flow for chemical intermediates

Data Analysis Methods:

• For simple Michaelis-Menten kinetics:

  • Direct linear plots

  • Non-linear regression (preferred)

  • Lineweaver-Burk plots (cautiously, for visualization only)

• For bisubstrate kinetics:

  • Global fitting of multiple datasets

  • Product inhibition studies

  • Isotope effects if applicable

Validation Approaches:

• Perform experiments under multiple conditions (pH, temperature, ionic strength)

• Use multiple data fitting methods and compare results

• Calculate standard errors and confidence intervals

• Validate with alternative assay methods when possible

Typical Kinetic Parameters from Related Enzymes:

How can I study the interaction between MCR1 and cytochrome b5 in Aspergillus clavatus?

Studying the interaction between MCR1 and cytochrome b5 in Aspergillus clavatus requires specialized techniques for protein-protein interactions:

Protein-Protein Interaction Assays:

• Co-immunoprecipitation (Co-IP)

  • Express tagged versions of both proteins

  • Precipitate one protein and detect the other by Western blotting

  • Controls should include individual proteins and non-specific interactions

• Surface Plasmon Resonance (SPR)

  • Immobilize purified MCR1 on a sensor chip

  • Flow cytochrome b5 solutions at different concentrations

  • Measure association and dissociation kinetics

  • Calculate binding affinity (KD)

• Isothermal Titration Calorimetry (ITC)

  • Directly measures thermodynamic parameters of binding

  • Provides enthalpy (ΔH), entropy (ΔS), and binding affinity (KD)

  • Requires significant amounts of purified proteins

Structural Analysis of the Complex:

• Cross-linking coupled with mass spectrometry

  • Use chemical cross-linkers to capture transient interactions

  • Digest cross-linked complexes and identify by mass spectrometry

  • Map interaction interfaces

• FRET (Förster Resonance Energy Transfer)

  • Label MCR1 and cytochrome b5 with compatible fluorophores

  • Measure energy transfer as indicator of proximity

  • Can be performed in vitro or in vivo with fluorescent protein fusions

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS)

  • Identifies regions protected from solvent upon complex formation

  • Maps interaction interfaces with high resolution

  • Requires specialized equipment and analysis

Computational Approaches:

• Molecular Docking

  • Generate homology models of both proteins if structures unavailable

  • Perform docking simulations to predict binding modes

  • Validate predictions with mutagenesis studies

• Molecular Dynamics Simulations

  • Simulate the dynamic behavior of the complex

  • Identify stable interactions and conformational changes

  • Calculate binding free energies

Functional Validation:

• Site-directed mutagenesis of predicted interface residues

• Kinetic analysis of mutants to correlate structure with function

• In vivo co-localization studies using fluorescently tagged proteins

How can recombinant Aspergillus clavatus MCR1 be used in biotechnological applications?

Recombinant Aspergillus clavatus MCR1 has several potential biotechnological applications:

Biocatalysis Applications:

• NADH Regeneration Systems:

  • MCR1 can be coupled with other oxidoreductases in biocatalytic cascades

  • Enables continuous regeneration of NADH for other enzymatic reactions

  • Particularly useful in fine chemical and pharmaceutical synthesis

• Redox Biotransformations:

  • MCR1 can participate in electron transfer chains for specific redox reactions

  • Applications in stereoselective reductions

  • Potential for biosensor development based on electron transfer capabilities

Pharmaceutical Research:

• Drug Target Exploration:

  • As fungi-specific enzymes, MCR1 and related proteins may serve as targets for antifungal development

  • High-throughput screening systems using recombinant MCR1 can identify potential inhibitors

  • Structure-based drug design approaches using MCR1 structural information

• Drug Metabolism Studies:

  • Cytochrome b5 reductases participate in drug metabolism pathways

  • Recombinant MCR1 can be used to study metabolism of xenobiotics

  • Comparative studies with human cytochrome b5 reductase for drug interaction prediction

Biosensor Development:

• Electrochemical Biosensors:

  • Immobilize MCR1 on electrode surfaces

  • Direct electron transfer between enzyme and electrode

  • Applications in NADH/NAD+ ratio monitoring

  • Potential for development of field-deployable sensors

• Optical Biosensors:

  • Utilize the spectral properties of the flavin cofactor

  • Monitor redox state changes in response to analytes

  • Develop fluorescence-based detection systems

Research Considerations:

• Enzyme stability must be optimized for commercial applications

• Immobilization strategies need to be developed for reusability

• Specific activity and substrate specificity may need engineering for particular applications

What genomic approaches can be used to study the evolution of MCR1 across Aspergillus species?

Studying the evolution of MCR1 across Aspergillus species requires sophisticated genomic approaches:

Comparative Genomics:

• Whole Genome Sequencing and Assembly:

  • Generate high-quality genome assemblies of multiple Aspergillus species

  • Ensure adequate coverage (>30x) for accurate gene prediction

  • Use both short and long-read technologies for optimal assembly

• Ortholog Identification:

  • Identify MCR1 orthologs across all sequenced Aspergillus species

  • Use reciprocal BLAST, OrthoMCL, or OrthoFinder for accurate ortholog assignment

  • Distinguish true orthologs from paralogs within gene families

• Synteny Analysis:

  • Examine conservation of gene order around MCR1 locus

  • Identify genomic rearrangements that may affect regulation

  • Map chromosomal location changes across species

Evolutionary Analysis:

• Phylogenetic Tree Construction:

  • Align MCR1 sequences using MUSCLE or MAFFT

  • Construct maximum likelihood or Bayesian phylogenetic trees

  • Compare gene trees with species trees to identify discordances

• Selection Pressure Analysis:

  • Calculate dN/dS ratios to detect positive or purifying selection

  • Use methods like PAML, HyPhy, or MEME to identify sites under selection

  • Correlate selection patterns with functional domains and binding sites

• Ancestral Sequence Reconstruction:

  • Infer ancestral MCR1 sequences at internal nodes of the phylogeny

  • Compare biochemical properties of ancestral vs. extant enzymes

  • Identify key evolutionary transitions in enzyme function

Population Genomics:

• Strain-Level Variation:

  • Sequence MCR1 from multiple strains within each Aspergillus species

  • Characterize intraspecific variation compared to interspecific differences

  • Identify potential adaptive polymorphisms

• Clade Analysis:

  • As seen in A. fumigatus population studies, distinct clades may exist with differential properties

  • Determine if MCR1 variation correlates with these population structures

  • Look for evidence of selective sweeps or balancing selection

Experimental Validation:

• Resurrection of Ancestral Proteins:

  • Synthesize and express reconstructed ancestral MCR1 sequences

  • Compare biochemical properties with extant enzymes

  • Test hypotheses about functional evolution

• Domain Swapping Experiments:

  • Create chimeric proteins with domains from different species

  • Identify domains responsible for species-specific properties

  • Map functional divergence to specific sequence changes

How can I design experiments to investigate the role of MCR1 in oxidative stress responses in Aspergillus clavatus?

Designing experiments to investigate MCR1's role in oxidative stress responses requires a comprehensive approach:

Genetic Manipulation Strategies:

• Generate MCR1 Mutant Strains:

  • Knockout mutants (ΔMCR1) using CRISPR-Cas9 or homologous recombination

  • Overexpression strains with constitutive or inducible promoters

  • Point mutants targeting catalytic residues or regulatory sites

• Create Reporter Strains:

  • Fluorescent protein fusions to monitor MCR1 localization

  • Promoter-reporter constructs to monitor MCR1 expression

  • Oxidative stress-responsive promoters driving reporters

Oxidative Stress Challenge Experiments:

• Chemical Stressors:

  • Hydrogen peroxide (H₂O₂): 0.1-10 mM

  • Menadione: 10-100 μM (superoxide generator)

  • tert-Butylhydroperoxide: 0.1-1 mM

  • Antimycin A: 1-10 μM (mitochondrial superoxide generator)

• Environmental Stressors:

  • Hypoxia/reoxygenation cycles

  • Temperature stress (heat shock)

  • UV radiation exposure

  • Antifungal drug exposure

Cellular Response Analysis:

• Viability and Growth Assays:

  • Survival curves following oxidative challenge

  • Growth rate measurements under stress conditions

  • Colony morphology assessment

  • Microscopic analysis of cellular morphology

• Biochemical Markers:

  • Intracellular ROS levels (DCFH-DA, CellROX, or MitoSOX staining)

  • Lipid peroxidation (TBARS assay, 4-HNE detection)

  • Protein carbonylation

  • Glutathione levels (GSH/GSSG ratio)

• Enzymatic Defense Systems:

  • Superoxide dismutase (SOD) activity

  • Catalase activity

  • Glutathione peroxidase activity

  • Comparative analysis between wild-type and MCR1 mutants

Molecular and Omics Approaches:

• Transcriptomics:

  • RNA-seq comparing wild-type and MCR1 mutants under stress

  • Time-course analysis of transcriptional responses

  • Identification of co-regulated gene networks

• Proteomics:

  • Global protein expression changes

  • Post-translational modifications (especially oxidative modifications)

  • Protein-protein interaction networks involving MCR1

• Metabolomics:

  • Redox metabolite profiling

  • NAD+/NADH and NADP+/NADPH ratios

  • Changes in mitochondrial metabolites

Physiological Measurements:

• Mitochondrial Function:

  • Oxygen consumption rates

  • Membrane potential measurements

  • ATP production capacity

  • Electron transport chain complex activities

• Redox Homeostasis:

  • Real-time monitoring of cellular redox state

  • Compartment-specific redox sensors

  • Electron paramagnetic resonance (EPR) spectroscopy

What are the methodological challenges in studying substrate specificity of Aspergillus clavatus MCR1?

Studying substrate specificity of Aspergillus clavatus MCR1 presents several methodological challenges that researchers should be aware of:

Protein Production Challenges:

• Heterologous Expression Issues:

  • Fungal proteins may have codon usage bias in bacterial systems

  • Proper folding and flavin incorporation may be incomplete

  • Expression levels may be low or protein may form inclusion bodies

• Protein Stability Concerns:

  • The enzyme may have limited stability after purification

  • Activity loss during storage and handling

  • Requirements for specific buffer components or additives

Assay Development Challenges:

• Electron Donor Specificity:

  • While NADH is the physiological electron donor, testing alternative donors requires:

    • Spectral overlap considerations in absorbance-based assays

    • Development of specific assay conditions for each donor

    • Correction for non-enzymatic reactions

• Electron Acceptor Specificity:

  • Cytochrome b5 is the natural acceptor, but testing others requires:

    • Purification of diverse electron acceptors

    • Development of specific detection methods for each acceptor

    • Consideration of direct electron transfer between NADH and acceptors

• Kinetic Analysis Complexities:

  • Bisubstrate reaction mechanisms complicate kinetic analysis

  • Product inhibition effects may distort kinetic parameters

  • Allosteric effects may lead to non-Michaelis-Menten behavior

Technical Measurement Challenges:

• Spectrophotometric Interference:

  • Absorption spectra of the enzyme, substrates, and products may overlap

  • Turbidity issues with membrane-associated substrates

  • Background oxidation of NADH in aerobic conditions

• Low Activity with Non-Preferred Substrates:

  • Detection limits for assays with poor substrates

  • Distinguishing genuine low activity from experimental noise

  • Need for highly sensitive detection methods (fluorescence, radioisotopes)

Physiological Relevance Challenges:

• In Vitro vs. In Vivo Correlation:

  • Substrate concentrations in vitro may not reflect cellular conditions

  • Cellular compartmentalization may restrict access to certain substrates

  • Protein-protein interactions may modify specificity in vivo

• Functional Redundancy:

  • Other reductases may compensate for MCR1 in knockout studies

  • Phenotypic effects may be subtle or condition-dependent

  • Need for multiple complementary approaches to confirm specificity

Methodological Solutions:

• Develop enzyme-coupled assay systems for challenging substrates

• Use recombinant cytochrome b5 from the same organism as preferred acceptor

• Employ stopped-flow techniques for transient kinetic analysis

• Validate in vitro findings with in vivo metabolic labeling studies

• Compare results across multiple experimental platforms and conditions