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

What is NADH-cytochrome b5 reductase 2 (mcr1) and what is its role in Aspergillus terreus?

NADH-cytochrome b5 reductase 2 (mcr1) is a critical enzyme in Aspergillus terreus that catalyzes the reduction of cytochrome b5 molecules using NADH as the physiological electron donor. The enzyme functions as part of the electron transfer chain in this pathogenic fungus. In its recombinant form, the protein is characterized by specific amino acid sequences including MFARQTFRYAQPLKQSFRKYSTEAPKGKSLAPVYLTVGLAGLGVGLYRYNSATAEAPAER and subsequent chain segments . The enzyme is encoded in Aspergillus terreus strain NIH 2624/FGSC A1156 and has been assigned UniProt number Q0CRD8 . As a flavoprotein, it plays essential roles in various metabolic pathways within the fungus, particularly in redox reactions necessary for cellular function.

How does the structure of Aspergillus terreus mcr1 compare to other cytochrome b5 reductases?

While specific structural information for Aspergillus terreus mcr1 is limited in the provided data, comparative analysis can be drawn from related cytochrome b5 reductases. Mammalian cb5r enzymes have been studied in greater detail, revealing critical structural elements likely conserved in fungal homologs. For instance, rat cytochrome b5 reductase has been crystallized at 2.0 Å resolution, with structures determined both alone and in complex with NAD+ .

The enzyme typically consists of two domains: an FAD-binding domain and an NADH-binding domain. Key catalytic residues in mammalian cb5r include specific lysine residues that play crucial roles in stabilizing the NADH-bound form of the enzyme . The active site architecture likely contains conserved residues for flavin binding and electron transfer, though specific differences in fungal reductases would affect substrate specificity and catalytic efficiency. Structural studies of the Aspergillus terreus enzyme would be necessary to determine precise structural homology with other species' reductases.

What are the optimal storage and handling conditions for recombinant Aspergillus terreus mcr1?

For optimal preservation of recombinant Aspergillus terreus NADH-cytochrome b5 reductase 2 (mcr1), the protein should be stored in a Tris-based buffer with 50% glycerol at -20°C for regular storage periods . For extended storage, preservation at -80°C is recommended to minimize protein degradation and maintain enzyme activity . Working aliquots should be kept at 4°C and used within one week to ensure consistency in experimental results .

Researchers should avoid repeated freeze-thaw cycles as these significantly reduce enzyme activity through denaturation and aggregation . It is advisable to prepare small working aliquots during initial receipt of the enzyme to minimize the need for repeated thawing of the stock solution. The protein should be handled according to standard laboratory practices for recombinant proteins, including using sterile techniques and appropriate personal protective equipment to prevent contamination.

What are the best methodological approaches for expressing and purifying recombinant Aspergillus terreus mcr1?

Recombinant expression of Aspergillus terreus NADH-cytochrome b5 reductase 2 can be accomplished using several expression systems, with E. coli being the most common for initial studies. For optimal expression, the following methodological approach is recommended:

• Gene optimization and vector design: The mcr1 coding sequence should be codon-optimized for the expression host. Inclusion of appropriate affinity tags (His-tag or GST-tag) facilitates downstream purification. The tag type may be determined during the production process to optimize protein yield and activity .

• Expression conditions: For E. coli expression systems, induction with IPTG at lower temperatures (16-20°C) often yields better results for fungal proteins by reducing inclusion body formation. Post-induction expression should proceed for 16-20 hours.

• Lysis and initial purification: Cell lysis should be performed in a Tris-based buffer (pH 7.5-8.0) containing protease inhibitors. Initial clarification through centrifugation at 15,000 × g for 30 minutes removes cellular debris.

• Affinity chromatography: Utilizing the affinity tag, the protein can be purified using appropriate resins. For His-tagged proteins, immobilized metal affinity chromatography with nickel or cobalt resins is effective.

• Secondary purification: Size-exclusion chromatography further purifies the protein and confirms its oligomeric state. For mcr1, which typically exists as a monomer, a Superdex 75 or 200 column is appropriate.

• Quality control: SDS-PAGE, western blotting, and activity assays should be performed to confirm identity, purity, and functionality.

The purified protein should be stored in a Tris-based buffer with 50% glycerol to maintain stability during storage .

How can researchers effectively measure the enzymatic activity of recombinant Aspergillus terreus mcr1?

Measuring the enzymatic activity of recombinant Aspergillus terreus NADH-cytochrome b5 reductase 2 requires assessment of its ability to transfer electrons from NADH to cytochrome b5. The following methodological approach provides a reliable measurement:

• Spectrophotometric assay: The most common method utilizes the change in absorbance at 340 nm as NADH (which absorbs at this wavelength) is oxidized to NAD+. Alternatively, using artificial electron acceptors like ferricyanide or dichlorophenolindophenol (DCIP) allows monitoring at different wavelengths.

• Reaction mixture composition:

  • 50 mM potassium phosphate buffer (pH 7.0-7.5)

  • 0.1 mM EDTA

  • 0.1 mM NADH

  • Appropriate concentration of cytochrome b5 (typically 10-50 μM)

  • Purified recombinant mcr1 enzyme (1-10 μg/mL)

• Data analysis: Calculate the initial reaction velocity (V₀) from the linear portion of the progress curve. Enzyme kinetic parameters (Km, kcat) can be determined using varying concentrations of NADH and cytochrome b5.

• Charge transfer assay: For more detailed kinetic analysis, a charge transfer assay can be employed to assess the efficiency of NADH utilization, similar to methods used for studying mammalian cb5r . This approach is particularly valuable for evaluating the effects of mutations or inhibitors on enzyme function.

• Controls and validation: Include appropriate negative controls (reaction mixture without enzyme) and positive controls (commercial cytochrome b5 reductase) to validate the assay system.

Temperature and pH optimization should be performed to determine optimal reaction conditions for the Aspergillus terreus enzyme, as these may differ from mammalian counterparts.

What structural and functional studies can be performed to identify critical catalytic residues in Aspergillus terreus mcr1?

Advanced structural and functional studies of Aspergillus terreus NADH-cytochrome b5 reductase 2 can reveal critical catalytic residues through a multi-faceted approach:

• X-ray crystallography: Obtaining high-resolution crystal structures of mcr1 both in its apo form and in complex with NAD+ would provide detailed insights into the active site architecture. Previous studies with rat cb5r achieved 2.0 Å resolution for the apo enzyme and 2.3 Å resolution for the NAD+ complex , serving as a methodological template.

• Site-directed mutagenesis: Based on sequence homology with characterized cb5r enzymes, potential catalytic residues can be identified and mutated. For example, in mammalian cb5r, lysine residues play crucial roles in NADH binding and catalysis . Systematic mutation of conserved residues in Aspergillus terreus mcr1 followed by kinetic analysis would identify functionally important amino acids.

• Enzyme kinetics with mutants: Measuring kinetic parameters (kcat, Km) for wild-type and mutant proteins allows quantification of each residue's contribution to catalysis. Specifically examining apparent Km values for NADH and cytochrome b5 separately can distinguish between effects on substrate binding versus catalytic efficiency .

• Molecular dynamics simulations: Computational methods can model protein dynamics and predict how mutations might affect enzyme flexibility, substrate binding, and catalysis. These simulations can guide experimental designs and help interpret experimental results.

• Charge transfer assays: Specialized assays measuring the charge transfer complex formation between the enzyme and NADH can detect subtle effects of mutations on NADH utilization efficiency .

Results from these studies could be compiled into a comprehensive table:

This comprehensive approach would establish structure-function relationships specific to the fungal enzyme and potentially identify targets for selective inhibition.

How can researchers develop selective inhibitors targeting Aspergillus terreus mcr1 as potential antifungal agents?

Developing selective inhibitors against Aspergillus terreus NADH-cytochrome b5 reductase 2 requires a systematic approach combining structural biology, medicinal chemistry, and microbiological validation:

• Structural basis for selectivity: Comparative structural analysis between fungal mcr1 and human cytochrome b5 reductase is essential to identify unique structural features in the fungal enzyme. X-ray crystallography or homology modeling can identify binding pockets present in the fungal enzyme but absent or structurally different in the human homolog.

• Virtual screening and rational design: Using the identified structural differences, virtual screening of compound libraries can identify potential hit compounds. Structure-based drug design approaches can then optimize these hits for increased potency and selectivity.

• Biochemical validation pipeline:

  • Primary screening: Inhibition assays using recombinant enzymes

  • Selectivity profiling: Testing against human cb5r and other related enzymes

  • Mode of inhibition studies: Determining competitive, non-competitive, or uncompetitive mechanisms

  • Structure-activity relationship development: Systematic modification of lead compounds

• Cellular validation: Promising inhibitors should be tested for:

  • Antifungal activity against Aspergillus terreus cultures

  • Cytotoxicity against human cell lines to assess safety

  • Ability to penetrate fungal cell walls

  • Effects on fungal metabolism beyond direct enzyme inhibition

• In vivo efficacy: Testing in appropriate animal models of Aspergillus infection to determine pharmacokinetics, efficacy, and safety

This approach leverages the molecular differences between fungal and human enzymes, potentially addressing the therapeutic challenge posed by Aspergillus terreus as an emerging pathogen affecting immunocompromised patients . The need for species-specific identification in clinical settings further emphasizes the value of targeted approaches based on unique fungal proteins .

What is the relationship between Aspergillus terreus mcr1 and fungal pathogenicity in immunocompromised patients?

The relationship between Aspergillus terreus NADH-cytochrome b5 reductase 2 and fungal pathogenicity in immunocompromised patients involves complex metabolic and virulence factors:

Understanding this relationship could lead to dual-purpose research outcomes: identification of new therapeutic targets and development of improved diagnostic methods for this emerging pathogen.

What are common technical challenges when working with recombinant Aspergillus terreus mcr1 and how can they be overcome?

Researchers working with recombinant Aspergillus terreus NADH-cytochrome b5 reductase 2 may encounter several technical challenges that can be addressed through methodological adjustments:

• Low expression yields:

  • Challenge: Fungal proteins often express poorly in bacterial systems

  • Solution: Optimize codon usage for the expression host, lower induction temperature to 16-18°C, use specialized E. coli strains (Rosetta, Arctic Express) designed for challenging proteins, or switch to eukaryotic expression systems like Pichia pastoris for better post-translational processing

• Protein insolubility and inclusion body formation:

  • Challenge: Recombinant mcr1 may form inclusion bodies

  • Solution: Add solubility tags (SUMO, MBP, TRX), optimize buffer conditions with stabilizing additives (glycerol, sucrose, specific detergents), or develop controlled refolding protocols if extraction from inclusion bodies is necessary

• Loss of FAD cofactor during purification:

  • Challenge: The FAD cofactor may dissociate during purification

  • Solution: Supplement buffers with FAD (1-10 μM) throughout the purification process, avoid harsh elution conditions, and confirm flavin content spectrophotometrically (A450/A275 ratio)

• Enzyme instability and activity loss:

  • Challenge: Rapid activity loss after purification

  • Solution: Optimize storage buffer with stabilizers (50% glycerol is recommended) , avoid repeated freeze-thaw cycles , prepare small working aliquots, and add reducing agents (DTT or β-mercaptoethanol) to prevent oxidation of critical cysteine residues

• Inconsistent activity measurements:

  • Challenge: Variable results in enzyme assays

  • Solution: Standardize cytochrome b5 source and concentration, ensure NADH quality (prepare fresh solutions), control temperature precisely during assays, and include internal standards to normalize between experiments

Implementing these methodological adjustments can significantly improve research outcomes when working with this challenging fungal enzyme.

How can researchers differentiate between Aspergillus terreus mcr1 and other mcr-related genes or proteins in experimental contexts?

Differentiating Aspergillus terreus NADH-cytochrome b5 reductase 2 (mcr1) from other mcr-related genes or proteins requires careful methodological approaches to avoid confusion, particularly with bacterial mcr-1 associated with colistin resistance:

• Sequence-based differentiation:

  • Perform thorough phylogenetic analysis comparing the Aspergillus terreus mcr1 sequence (UniProt: Q0CRD8) with bacterial mcr-1 genes (associated with colistin resistance) and human MCR-related genes

  • Design PCR primers targeting unique regions of the fungal gene that show minimal homology with bacterial or human counterparts

  • Implement specific hybridization conditions in Southern or Northern blotting to ensure target specificity

• Protein-based identification:

  • Develop specific antibodies that recognize epitopes unique to Aspergillus terreus mcr1

  • Employ Western blotting with careful controls to confirm specificity

  • Use mass spectrometry to identify unique peptide fragments that differentiate between fungal mcr1 and other mcr proteins

• Functional discrimination:

  • Aspergillus terreus mcr1 functions as an NADH-dependent reductase, while bacterial mcr-1 functions in phosphoethanolamine transfer in lipopolysaccharide modification

  • Establish activity assays that distinguish between these fundamentally different functions

  • For Aspergillus terreus mcr1, measure NADH oxidation coupled to cytochrome b5 reduction

  • For bacterial mcr-1, assess phosphoethanolamine transferase activity

• Experimental design considerations:

  • When publishing, clearly define abbreviations and provide full protein names to avoid confusion

  • Include appropriate positive and negative controls in all experiments

  • When working with clinical samples that might contain both fungal and bacterial components, employ selective cultivation methods or species-specific molecular detection techniques

This methodological approach prevents confusion between the fungal reductase mcr1 and bacterial colistin resistance genes (mcr-1, mcr-2, etc.), which represent entirely different protein families despite similar abbreviations in the literature .

What are promising research avenues for understanding the role of Aspergillus terreus mcr1 in fungal metabolism and drug resistance?

Future research on Aspergillus terreus NADH-cytochrome b5 reductase 2 (mcr1) presents several promising avenues for understanding both fundamental fungal biology and potential clinical applications:

• Systems biology approach to metabolic networks:

  • Integrate mcr1 function into genome-scale metabolic models of A. terreus

  • Employ flux balance analysis to predict metabolic shifts under different environmental conditions

  • Use metabolomic profiling to identify downstream metabolites affected by mcr1 activity

  • This approach would clarify how electron transfer systems contribute to fungal adaptability in host environments

• Drug resistance connections:

  • Investigate potential links between mcr1 activity and resistance to current antifungal agents

  • Examine whether upregulation of mcr1 occurs in response to azole or echinocandin exposure

  • Determine if mcr1 contributes to detoxification pathways that might reduce antifungal efficacy

  • Study potential synergistic effects between mcr1 inhibitors and existing antifungals

• Comparative genomics and protein evolution:

  • Compare mcr1 sequences across pathogenic and non-pathogenic Aspergillus species

  • Identify evolutionary adaptations in mcr1 that might correlate with pathogenicity

  • Analyze selection pressures on mcr1 in clinical versus environmental isolates

  • This evolutionary perspective could reveal how redox systems adapt during host-pathogen coevolution

• Development of nanoantibody-based detection systems:

  • Building on previous work with monoclonal antibodies to Aspergillus proteins

  • Design nanoantibodies targeting unique epitopes of mcr1

  • Develop rapid diagnostic platforms for species-specific identification

  • Explore theranostic approaches combining detection and targeted inhibition

These research directions could significantly advance our understanding of A. terreus as an emerging pathogen affecting immunocompromised patients while potentially yielding new diagnostic and therapeutic strategies.

How might structural comparisons between fungal and human cytochrome b5 reductases inform targeted drug development?

Structural comparisons between fungal and human cytochrome b5 reductases represent a critical approach for developing targeted antifungal agents with minimal host toxicity:

• Comparative structural biology approach:

  • Determine high-resolution crystal structures of Aspergillus terreus mcr1 in parallel with human cytochrome b5 reductase

  • Create detailed binding site maps highlighting conserved versus divergent regions

  • Identify unique structural features in the fungal enzyme that could serve as selective targets

  • Analysis of the rat cb5r structure at 2.0 Å resolution provides methodological guidance for this approach

• Active site architecture analysis:

  • Compare catalytic residues between fungal and human enzymes to identify subtle differences in spatial arrangement

  • Map electrostatic surface potentials to identify differential charge distributions that might be exploited for selective binding

  • Analyze cofactor binding pockets, as even minor differences can be leveraged for selective inhibitor design

  • Previous work identifying the importance of specific lysine residues in mammalian cb5r provides a foundation for such comparisons

• Dynamic structural elements:

  • Employ molecular dynamics simulations to identify differences in protein flexibility and conformational changes

  • Analyze protein-protein interaction interfaces, particularly with cytochrome b5

  • Identify allosteric sites unique to the fungal enzyme that could be targeted for selective inhibition

• Drug design strategy based on structural insights:

  • Focus on binding pockets present only in the fungal enzyme

  • Design inhibitors that exploit unique structural features

  • Develop compounds that preferentially interact with fungal-specific residues

  • Implement structure-based virtual screening to identify lead compounds

This structure-based approach directly addresses the need for selective antifungal agents against Aspergillus terreus, an emerging pathogen affecting immunocompromised patients .