Recombinant Uncharacterized oxidoreductase MAP_4149 (MAP_4149)

What is Uncharacterized oxidoreductase MAP_4149 and what organism does it originate from?

MAP_4149 is an uncharacterized oxidoreductase protein from Mycobacterium paratuberculosis, a member of the mycobacteria group. The protein encompasses amino acids 1-286 and belongs to the oxidoreductase enzyme family, which catalyzes oxidation-reduction reactions. Despite being identified in the M. paratuberculosis genome, its specific biological function remains largely uncharacterized, hence the designation as "uncharacterized oxidoreductase" .

Mycobacterium paratuberculosis is related to other mycobacteria like M. tuberculosis (the causative agent of tuberculosis), but has its own distinct characteristics. Understanding MAP_4149 may provide insights into the metabolism and pathogenicity of this organism, potentially contributing to research on mycobacterial infections and their treatment strategies .

What expression systems are commonly used for producing recombinant MAP_4149?

For expression of recombinant MAP_4149, researchers can employ several established expression systems, each with specific advantages:

The choice depends on research objectives - E. coli systems are often preferred for initial characterization due to rapid turnaround and high yields, while mammalian expression may be necessary when authentic folding and modifications are critical for functional studies .

What are the optimal storage conditions for recombinant MAP_4149?

While specific storage conditions for MAP_4149 are not explicitly detailed in the available literature, oxidoreductase proteins typically require careful storage to maintain enzymatic activity. The recommended protocol generally includes:

• Short-term storage (1-2 weeks): 4°C in appropriate buffer systems with stabilizing agents

• Medium-term storage (1-6 months): -20°C with cryoprotectants such as glycerol (20-50%)

• Long-term storage: -80°C in aliquots to avoid freeze-thaw cycles

Enzyme activity should be periodically verified, especially after extended storage periods, as oxidoreductases can lose functionality through oxidation of critical residues or structural changes .

How can I design experiments to characterize the enzymatic activity of MAP_4149?

To characterize the enzymatic activity of this uncharacterized oxidoreductase, a systematic approach is recommended:

• Substrate screening: Test potential oxidoreductase substrates using a panel of common electron donors (NADH, NADPH) and acceptors (various quinones, cytochromes)

• Optimal conditions determination:

  • pH range (typically 5.0-9.0 in 0.5 increments)

  • Temperature range (25-45°C)

  • Metal ion requirements (test with EDTA chelation and addition of Mg²⁺, Mn²⁺, Zn²⁺, Fe²⁺)

• Kinetic parameter measurement:

  • Determine Km and Vmax using varied substrate concentrations

  • Calculate kcat and catalytic efficiency (kcat/Km)

• Inhibition studies:

  • Test with class-specific oxidoreductase inhibitors

  • Analyze competitive vs. non-competitive inhibition patterns

Each experiment should include appropriate controls, including heat-inactivated enzyme and reactions without enzyme or substrate .

What purification strategies are most effective for recombinant MAP_4149?

Purification of recombinant MAP_4149 typically employs a multi-step approach to achieve high purity:

The purification protocol may require optimization based on the expression system used. For E. coli-expressed MAP_4149, inclusion body formation may necessitate refolding steps, while mammalian-expressed protein might require different initial capture strategies .

How can I assess the structural properties of MAP_4149?

Structural characterization of MAP_4149 can be approached through complementary techniques:

Combining these approaches provides comprehensive structural insights that can guide functional studies and potentially reveal catalytic mechanisms .

How might MAP_4149 compare functionally to characterized oxidoreductases in other mycobacterial species?

While MAP_4149 remains uncharacterized, comparative analysis with characterized oxidoreductases from related mycobacterial species can provide functional insights:

What is the potential role of MAP_4149 in Mycobacterium paratuberculosis pathogenicity?

The relationship between MAP_4149 and pathogenicity remains an open research question. Based on studies of oxidoreductases in other pathogenic mycobacteria, several hypotheses warrant investigation:

• Oxidative stress response: MAP_4149 may participate in detoxification of reactive oxygen species encountered during host immune response

• Metabolic adaptation: The enzyme could enable utilization of alternative electron donors/acceptors in nutrient-limited host environments

• Cell wall modification: Some mycobacterial oxidoreductases participate in cell wall component synthesis, affecting permeability and drug resistance

• Immunomodulation: Certain bacterial oxidoreductases have been shown to interact with host immune receptors, potentially altering inflammatory responses

Research using gene knockout or knockdown approaches, coupled with infection models, would help elucidate the role of MAP_4149 in virulence and pathogenesis .

How can protein-protein interaction studies inform MAP_4149 function?

Uncovering the interactome of MAP_4149 can provide significant insights into its biological context and function. Recommended approaches include:

• Affinity purification-mass spectrometry (AP-MS):

  • Express tagged MAP_4149 in mycobacterial cells

  • Capture protein complexes containing MAP_4149

  • Identify interaction partners by mass spectrometry

• Yeast two-hybrid screening:

  • Screen against M. paratuberculosis genomic library

  • Validate positive interactions with co-immunoprecipitation

• Proximity labeling techniques:

  • BioID or APEX2 fusion proteins to identify proximal proteins in living cells

  • Particularly useful for transient or context-dependent interactions

• Computational prediction:

  • Leverage structural data to predict potential interaction interfaces

  • Network analysis to identify functional clusters

Recent research on microtubule-associated protein 4 (MAP4, unrelated to MAP_4149) demonstrates how protein interaction studies can reveal significant functional insights, as MAP4 was found to interact with dynein and dynactin, affecting organelle transport .

What strategies can address low expression yields of recombinant MAP_4149?

When encountering low expression yields of recombinant MAP_4149, consider these systematic troubleshooting approaches:

• Expression vector optimization:

  • Test different promoters (T7, tac, AOX1 for yeast)

  • Optimize codon usage for the host organism

  • Include solubility-enhancing fusion tags (MBP, SUMO, Thioredoxin)

• Expression conditions optimization:

  • Temperature reduction (16-25°C) to slow folding

  • Inducer concentration titration

  • Culture media enrichment or specialized formulations

• Host strain selection:

  • For E. coli: BL21(DE3), Rosetta for rare codons, Origami for disulfide bonds

  • For yeast: Protease-deficient strains

• Co-expression strategies:

  • Molecular chaperones (GroEL/ES, DnaK)

  • Redox-modulating enzymes for oxidoreductases

Systematic testing and documentation of these variables will help identify optimal conditions for MAP_4149 expression .

How can researchers resolve issues with protein misfolding during recombinant MAP_4149 production?

Protein misfolding is a common challenge with recombinant oxidoreductases. These approaches may improve folding outcomes:

• In vivo folding enhancement:

  • Slow expression rate using lower temperatures and inducer concentrations

  • Co-express molecular chaperones specific to oxidoreductases

  • Include redox-balancing compounds in growth media

• Refolding from inclusion bodies:

  • Optimize solubilization conditions (8M urea or 6M guanidine HCl)

  • Test step-wise or rapid dilution refolding protocols

  • Include proper redox conditions (glutathione oxidized:reduced ratios)

  • Add stabilizing agents (arginine, glycerol, non-detergent sulfobetaines)

• Folding assessment methods:

  • Intrinsic fluorescence to monitor tertiary structure

  • Limited proteolysis to identify compact domains

  • Activity assays to confirm functional folding

A systematic refolding screen varying these parameters can identify optimal conditions for obtaining correctly folded MAP_4149 .

What analytical techniques are most suitable for confirming the identity and purity of recombinant MAP_4149?

Confirming the identity and purity of recombinant MAP_4149 requires multiple complementary analytical techniques:

For oxidoreductases like MAP_4149, additional specific metrics include:

• Enzymatic activity per mg protein (specific activity)

• A280/A260 ratio to detect nucleic acid contamination

• Metal content analysis if the enzyme requires cofactors

A purity level of >95% is typically required for detailed structural and functional characterization .

How might MAP_4149 be utilized in understanding mycobacterial metabolism?

MAP_4149, as an uncharacterized oxidoreductase, represents an opportunity to expand our understanding of mycobacterial metabolism through several research avenues:

• Metabolic pathway reconstruction:

  • Metabolomics studies comparing wild-type and MAP_4149 knockout strains

  • Isotope labeling to track substrates and identify the specific reactions catalyzed

  • Integration with existing metabolic models of mycobacteria

• Adaptation mechanisms:

  • Expression profiling under various growth conditions and stressors

  • Comparison of expression patterns in vitro versus in vivo during infection

  • Correlation with other metabolic enzyme activities

• Comparative genomics:

  • Analysis across pathogenic and non-pathogenic mycobacterial species

  • Evolutionary conservation patterns suggesting functional importance

  • Identification of species-specific adaptations

Understanding MAP_4149's role would contribute to the broader knowledge of how mycobacteria adapt their metabolism during infection and environmental stress, potentially revealing new therapeutic targets .

What are the implications of MAP_4149 research for drug discovery targeting mycobacterial infections?

Research on MAP_4149 has several potential implications for antimycobacterial drug discovery:

• Novel target validation:

  • Determine essentiality through gene knockout or CRISPRi approaches

  • Evaluate contribution to virulence in infection models

  • Assess conservation across mycobacterial pathogens

• Inhibitor development strategies:

  • Structure-based drug design once crystal structure is obtained

  • Fragment-based screening targeting the active site

  • Development of transition-state analogs specific to the catalyzed reaction

• Resistance mechanisms investigation:

  • Study of compensatory metabolic pathways

  • Natural variation in gene sequence across clinical isolates

  • Potential for acquired resistance through mutations

• Combination therapy opportunities:

  • Synergistic effects with existing antimycobacterials

  • Metabolic vulnerability exploitation in combination treatments

The unique biochemistry of mycobacterial oxidoreductases makes them attractive targets for selective inhibition with minimal host toxicity .

How can systems biology approaches enhance our understanding of MAP_4149 function?

Systems biology approaches offer powerful frameworks for elucidating MAP_4149 function within the broader context of mycobacterial biology:

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and metabolomics data from MAP_4149 perturbation studies

  • Identify regulatory networks and metabolic pathways affected by MAP_4149

  • Construct predictive models of enzyme function based on system-wide effects

• Network analysis:

  • Place MAP_4149 within protein-protein interaction networks

  • Identify functional modules and pathway connections

  • Compare network positioning with known oxidoreductases

• Flux balance analysis:

  • Incorporate MAP_4149 into genome-scale metabolic models

  • Predict metabolic flux distributions under various conditions

  • Validate predictions with experimental measurements

• Host-pathogen interaction modeling:

  • Study effects of MAP_4149 on host cell metabolism during infection

  • Model metabolic cross-talk between host and pathogen

  • Predict intervention points for disrupting infection progression

These integrative approaches can place the molecular function of MAP_4149 into a broader biological context, revealing its significance within mycobacterial physiology and pathogenesis .