Recombinant Candida glabrata Methylthioribulose-1-phosphate dehydratase (MDE1)

What expression systems are most effective for producing functional recombinant Candida glabrata MDE1?

Several expression systems can be employed for producing recombinant C. glabrata MDE1, each with specific advantages depending on your research objectives. Based on available data and standard practices in recombinant protein production, the following systems merit consideration:

For functional studies of C. glabrata MDE1, an E. coli or yeast-based expression system typically provides the best balance of yield and proper folding. The choice between N-terminal or C-terminal tags should be determined based on protein-tag stability factors . When expressed in E. coli, researchers should optimize induction conditions and consider lower temperatures (16-20°C) during induction to improve folding.

What are the optimal methods for measuring MDE1 enzymatic activity in vitro?

Measuring the enzymatic activity of recombinant C. glabrata MDE1 involves tracking the conversion of MTRu-1-P to DK-MTP-1-P. Based on established protocols for similar dehydratases, the following methodological approaches are recommended:

Direct Assay Approach:

• Substrate preparation: Chemically synthesize or enzymatically prepare MTRu-1-P as substrate

• Reaction conditions: Buffer at pH 7.5-8.5, temperature 25-40°C based on comparative data from B. subtilis enzyme

• Activity measurement: Monitor the disappearance of MTRu-1-P or appearance of DK-MTP-1-P

Spectrophotometric Coupled Assay:
Since the product DK-MTP-1-P is unstable (decomposing with a rate constant of 0.048 s⁻¹ in B. subtilis studies), a coupled enzyme assay may be more reliable . This would involve:

• Conversion of MTRu-1-P to DK-MTP-1-P by MDE1

• Immediate processing of DK-MTP-1-P by the next enzyme in the pathway (DK-MTP-1-P enolase)

• Monitoring the coupled reaction using spectrophotometric methods

Kinetic Parameters Determination:
For comprehensive kinetic analysis of the recombinant enzyme, researchers should determine:

• K₍ₘ₎ and V₍ₘₐₓ₎ values across a range of substrate concentrations

• pH and temperature optima

• Effects of potential inhibitors or activators

• Thermal stability profile

Researchers should note that when using these methods, it's crucial to account for the instability of the DK-MTP-1-P product, which can decompose to compounds not utilized by the next enzyme in the pathway .

How can researchers design effective MDE1 knockout or mutant studies in Candida glabrata?

For genetic manipulation studies targeting MDE1 in C. glabrata, researchers now have access to modern genetic tools that enable precise gene modifications. Based on current methodologies for C. glabrata:

CRISPR-Cas9 System Application:
C. glabrata genetic modification has been revolutionized by CRISPR-Cas9 technology. To apply this for MDE1 studies:

• Develop a recombinant strain of C. glabrata constitutively expressing the CRISPR-Cas9 system

• Use specialized online tools to select the most efficient guide RNAs targeting the MDE1 gene

• Identify mutant strains using Surveyor technique and sequencing

Deletion Library Resources:
Researchers can leverage existing C. glabrata deletion libraries that contain numerous bar-coded mutant strains. A comprehensive library containing 619 unique, individually bar-coded mutant strains representing approximately 12% of the genome is available, which may include MDE1 mutants .

Phenotypic Analysis Workflow:
After generating MDE1 knockout or mutant strains, perform these analyses:

• Growth phenotyping under normal conditions

• Stress response characterization

• Methionine utilization efficiency testing

• Comparative transcriptomics to identify compensatory pathways

• Virulence assessment using appropriate infection models, such as Drosophila melanogaster

For rigorous validation of mutant phenotypes, always include appropriate controls and complemented strains to confirm observed effects are directly attributable to MDE1 disruption rather than secondary genomic alterations.

What are the relationships between MDE1 and other metabolic pathways in Candida glabrata?

MDE1 functions within a complex metabolic network in C. glabrata, with connections to several critical pathways. Understanding these relationships is essential for comprehensive experimental design:

Integration with Regulatory Networks:
C. glabrata has evolved specific regulatory networks for adaptation to the human host environment . The methionine salvage pathway, where MDE1 functions, likely interfaces with these regulatory systems. Researchers should consider potential cross-regulation between:

• Sulfur metabolism control systems

• Stress response pathways

• Virulence factor regulation

Potential Connection to Drug Resistance Mechanisms:
C. glabrata exhibits inherent tolerance to azole drugs, mediated by transcription factors like CgPdr1 . While direct evidence linking MDE1 to drug resistance isn't provided in the search results, researchers should investigate potential metabolic links between:

• Methionine salvage pathway activity

• Cellular stress responses

• Expression of drug efflux pumps and other resistance mechanisms

Experimental Approaches to Study Pathway Integration:

• Metabolic flux analysis comparing wild-type and MDE1-deficient strains

• Transcriptomic profiling under varied methionine availability conditions

• Epistasis studies with regulatory genes known to control metabolism and virulence

• Proteomic analysis to identify physical interactions between MDE1 and other proteins

These approaches can reveal how MDE1 function is integrated into the broader metabolic landscape of C. glabrata and potentially identify novel regulatory mechanisms that coordinate metabolism with virulence and stress responses.

What are the optimal storage and handling conditions for maintaining recombinant MDE1 stability?

Proper storage and handling of recombinant C. glabrata MDE1 is critical for maintaining enzymatic activity. Based on standard protein biochemistry principles and specific information from the search results:

Storage Recommendations:

• For long-term storage: Store lyophilized protein at -20°C or -80°C

• Working aliquots: Store at 4°C for up to one week

• Avoid repeated freeze-thaw cycles as this significantly reduces activity

Reconstitution Protocol:

• Briefly centrifuge the product vial to ensure all material is at the bottom

• Reconstitute in appropriate buffer according to experimental requirements

• Mix gently to avoid foaming or denaturation

• Filter-sterilize if necessary for downstream applications

Stability Enhancement Factors:

• Consider adding stabilizers such as glycerol (10-20%) for frozen aliquots

• Inclusion of reducing agents may help maintain cysteine residues in reduced state

• Buffer systems should maintain optimal pH range (7.5-8.5) based on similar enzymes

Quality Control Monitoring:
Researchers should periodically check:

• Enzymatic activity using standardized assays

• Protein integrity via SDS-PAGE

• Aggregation status via dynamic light scattering or size exclusion chromatography

These handling practices will help ensure experimental reproducibility and maximize the functional lifespan of recombinant MDE1 preparations.

How can researchers effectively purify recombinant MDE1 while maintaining enzymatic activity?

Purification of recombinant C. glabrata MDE1 requires careful consideration of techniques that preserve enzymatic activity. Based on standard practices for recombinant enzymes and information from the search results:

Affinity Purification Strategy:
The recombinant MDE1 typically contains affinity tags to facilitate purification . Recommended approaches include:

• Immobilized metal affinity chromatography (IMAC) for His-tagged protein

• Glutathione affinity chromatography for GST-tagged protein

• Consider mild elution conditions to preserve activity

Purification Protocol Outline:

• Cell lysis: Gentle methods such as enzymatic lysis or mild sonication

• Clarification: Centrifugation at high speed (≥20,000 × g) to remove cell debris

• Affinity chromatography: Using appropriate resin based on tag system

• Optional secondary purification: Size exclusion or ion exchange chromatography

• Buffer exchange: Dialysis or gel filtration into storage buffer

Critical Factors for Activity Preservation:

• Temperature control: Maintain samples at 4°C throughout purification

• Protease inhibitors: Include comprehensive inhibitor cocktail

• Reducing environment: Consider adding DTT or β-mercaptoethanol

• pH maintenance: Buffer systems that maintain pH 7.5-8.5

Purity Assessment:
The target purity should be ≥85% as determined by SDS-PAGE . Higher purity (≥95%) may be required for crystallography or detailed kinetic studies.

What approaches can be used to investigate MDE1's role in Candida glabrata virulence?

Investigating MDE1's contribution to C. glabrata virulence requires multifaceted approaches that link enzymatic function to pathogenicity:

In Vitro Virulence Factor Assessment:

• Adhesion assays: Determine if MDE1 deletion affects adherence to host cells

• Biofilm formation: Quantify biofilm development capacity in wild-type vs. MDE1 mutants

• Stress resistance: Evaluate oxidative and nutrient stress responses

Infection Model Systems:
C. glabrata virulence can be assessed using various models, with these methodological considerations:

• Drosophila melanogaster model: Well-established for initial virulence screening

  • Advantages: Rapid, economical, ethical considerations

  • Measurement endpoints: Survival curves, fungal burden, host immune response

• Mammalian models: For more translatable virulence assessments

  • Mouse models of disseminated or mucosal candidiasis

  • Measurement endpoints: Organ fungal burden, inflammatory markers, survival

Regulatory Network Analysis:
MDE1 function may intersect with known virulence regulatory networks in C. glabrata:

• Investigate potential connections to CgPdr1-regulated genes

• Examine relationships with Mss11, which plays a crucial role in adhesion and biofilm formation

• Perform comparative transcriptomics between wild-type and MDE1-deficient strains during infection

Complementation Studies:
To establish causality, researchers should:

• Create complemented strains restoring MDE1 function

• Perform side-by-side comparisons of wild-type, knockout, and complemented strains

• Include heterologous complementation with MDE1 orthologs from related species

These approaches collectively provide comprehensive insights into how MDE1 function may contribute to C. glabrata's pathogenic potential.

How does C. glabrata MDE1 compare to orthologous enzymes in related species?

Comparative analysis of C. glabrata MDE1 with orthologous enzymes from related species provides evolutionary context and functional insights:

Phylogenetic Relationship Analysis:
C. glabrata is more closely related to Saccharomyces cerevisiae than to other Candida species like C. albicans, despite its classification as a Candida species . This evolutionary position is significant when comparing MDE1 orthologs:

• Comparison with S. cerevisiae ortholog offers insights into conserved functions

• Differences between C. glabrata and pathogenic Candida species may reveal pathogenicity-specific adaptations

• Comparison with environmental Nakaseomyces species helps identify human host adaptation features

Functional Conservation Assessment:
Based on studies of the MTRu-1-P dehydratase from B. subtilis, which catalyzes the same reaction, researchers can explore:

• Conservation of catalytic residues across species

• Substrate specificity differences

• Regulation mechanisms variation

• Kinetic parameter differences (the B. subtilis enzyme shows Kₘ of 8.9 μM and Vₘₐₓ of 42.7 μmol min⁻¹ mg⁻¹)

Structural Homology Considerations:
While specific structural data for C. glabrata MDE1 is limited in the search results, researchers can:

• Generate homology models based on available crystal structures

• Identify conserved domains and structural features

• Predict structure-function relationships that may be experimentally tested

This comparative approach helps contextualize C. glabrata MDE1 within evolutionary frameworks and provides hypotheses about potential functional specializations in this pathogenic yeast.

What methodological approaches are most effective for studying MDE1 interactions with other proteins?

Investigating protein-protein interactions involving MDE1 requires sophisticated methodological approaches:

Affinity-Based Interaction Screening:

• Co-immunoprecipitation (Co-IP): Using antibodies against tagged MDE1 to pull down interacting partners

• Tandem Affinity Purification (TAP): Leveraging the TAP-tag system already established for C. glabrata studies

• Proximity-dependent biotin labeling: BioID or TurboID approaches to identify proteins in close proximity to MDE1

Genetic Interaction Mapping:

• Synthetic genetic arrays: Crossing MDE1 mutants with genome-wide deletion collections

• Double-knockout analysis: Creating double mutants of MDE1 and candidate interactors

• Suppressor screens: Identifying mutations that rescue MDE1 deletion phenotypes

Advanced Biophysical Methods:

• Surface plasmon resonance (SPR): Measuring direct binding kinetics between MDE1 and candidate partners

• Isothermal titration calorimetry (ITC): Determining thermodynamic parameters of interactions

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Mapping interaction interfaces

In Silico Interaction Prediction:

• Leveraging existing interactome data from S. cerevisiae as a model

• Using computational tools to predict functional associations

• Network analysis to place MDE1 in the context of metabolic and signaling pathways

For C. glabrata specifically, researchers can build on existing methodologies used to study other proteins, such as the chromatin immunoprecipitation sequencing (ChIP-seq) approach used to identify genomic binding sites for the Pdr1 transcription factor .

What are emerging technologies that could advance MDE1 research in Candida glabrata?

Several cutting-edge technologies hold promise for advancing our understanding of MDE1 function and regulation in C. glabrata:

CRISPR-Based Technologies:
Beyond basic gene knockout, advanced CRISPR applications offer new research possibilities:

• CRISPRi/CRISPRa systems: For tunable repression or activation of MDE1 expression

• Base editing: For introducing specific point mutations without double-strand breaks

• Prime editing: For precise gene modifications without donor templates

Single-Cell Technologies:
Understanding cell-to-cell variability in MDE1 expression and function:

• Single-cell RNA-seq: Revealing expression heterogeneity within populations

• Single-cell proteomics: Detecting protein-level variations

• Microfluidics-based phenotyping: Assessing functional heterogeneity

Structural Biology Advances:
High-resolution structural insights into MDE1 function:

• Cryo-electron microscopy: For detailed structural analysis without crystallization

• AlphaFold2 and related AI tools: For highly accurate structural predictions

• Time-resolved structural methods: To capture enzyme dynamics during catalysis

Systems Biology Approaches:
Integrating MDE1 function into whole-cell models:

• Multi-omics integration: Combining transcriptomics, proteomics, and metabolomics data

• Genome-scale metabolic modeling: Predicting effects of MDE1 perturbation on cellular metabolism

• Regulatory network reconstruction: Identifying control mechanisms governing MDE1 expression

These emerging technologies will enable researchers to address more sophisticated questions about MDE1 function in C. glabrata and potentially identify novel intervention strategies targeting this enzyme or its associated pathways.

How might MDE1 serve as a potential target for antifungal development against Candida glabrata?

The exploration of MDE1 as a potential antifungal target requires systematic investigation of several key aspects:

Target Validation Framework:

• Essentiality assessment: Determine whether MDE1 is essential for C. glabrata survival in relevant host environments

• Virulence contribution: Quantify the impact of MDE1 deletion on virulence in infection models

• Metabolic bottleneck analysis: Evaluate whether MDE1 inhibition creates metabolic vulnerabilities

Structural and Functional Considerations for Inhibitor Design:

• Active site mapping: Identify catalytic residues and substrate binding pockets

• Species selectivity: Compare fungal MDE1 structure with any human homologs to ensure specificity

• Allosteric regulation sites: Explore potential for targeting non-active site regulatory domains

High-Throughput Screening Strategies:

• In vitro enzyme assays: Develop robust biochemical screens for inhibitor discovery

• Whole-cell phenotypic screens: Identify compounds that phenocopy MDE1 deletion

• Fragment-based drug discovery: Build inhibitors iteratively from small chemical fragments

Combination Therapy Potential:

• Synergy testing: Evaluate whether MDE1 inhibition sensitizes C. glabrata to existing antifungals

• Resistance mechanisms: Assess potential for resistance development against MDE1 inhibitors

• Multi-target approaches: Consider dual-action compounds targeting MDE1 and related pathways

Given C. glabrata's inherent tolerance to azole drugs , targeting metabolic enzymes like MDE1 represents a promising alternative strategy that could circumvent existing resistance mechanisms and expand the therapeutic arsenal against this important fungal pathogen.