Recombinant Candida glabrata Ribulose-phosphate 3-epimerase (RPE1)

What is Ribulose-phosphate 3-epimerase (RPE1) and what role does it play in Candida glabrata metabolism?

Ribulose-phosphate 3-epimerase (RPE1) is a critical enzyme involved in both the pentose phosphate pathway (PPP) and the Calvin-Benson-Bassham (CBB) cycle. In organisms like Candida glabrata, RPE1 catalyzes the reversible epimerization between ribulose-5-phosphate (Ru5P) and xylulose-5-phosphate (X5P). This reaction is essential for carbohydrate metabolism and contributes to both catabolic processes and biosynthetic pathways .

Similar to other eukaryotic microorganisms, C. glabrata likely utilizes RPE1 primarily in the PPP, which generates NADPH for reductive biosynthesis and pentoses for nucleotide synthesis. Unlike photosynthetic organisms where RPE also participates in the CBB cycle for carbon fixation, C. glabrata as a non-photosynthetic fungus would employ RPE1 exclusively in the PPP for sugar metabolism and redox balance maintenance.

How does Candida glabrata RPE1 differ structurally and functionally from its homologs in other species?

While specific structural data for Candida glabrata RPE1 is limited, comparative analysis can be inferred from studies on homologous enzymes. RPEs typically display a conserved homo-hexameric quaternary structure with a triose isomerase-type (TIM-) barrel fold that contains an α8β8 structure, exposing catalytic pockets on the top of each barrel .

In terms of functional differences, RPE1 enzymes from different species exhibit varied substrate affinities and catalytic efficiencies. For instance, the Chlamydomonas reinhardtii RPE1 demonstrates higher affinity for X5P (KM = 0.716 ± 0.09 mM) compared to Ru5P while displaying greater catalytic efficiency with Ru5P as a substrate . C. glabrata RPE1 likely exhibits species-specific kinetic parameters adapted to its metabolic requirements as a human pathogen, potentially influencing its virulence and survival within host environments.

What are the recommended methods for cloning and expressing recombinant Candida glabrata RPE1?

For successful cloning and expression of recombinant C. glabrata RPE1, researchers should consider the following methodological approach:

• Gene identification and primer design: The RPE1 gene should be identified in the C. glabrata genome using bioinformatic tools. Design PCR primers with appropriate restriction sites for directional cloning.

• Expression vector selection: Choose a bacterial expression system (typically E. coli BL21(DE3) or similar strains) with appropriate fusion tags for purification. His-tagged constructs are commonly used for metal affinity chromatography.

• Optimization of expression conditions: Systematic testing of induction conditions including temperature (typically 16-25°C for fungal proteins), IPTG concentration (0.1-1.0 mM), and induction time (4-18 hours) to maximize soluble protein yield.

• Cell lysis and protein extraction: Mechanical disruption combined with gentle detergents to preserve enzymatic activity, followed by centrifugation to separate soluble fractions.

When adapting protocols from homologous enzymes like CrRPE1, researchers should account for potential differences in codon usage bias, protein solubility, and post-translational modifications between algal and fungal systems .

What purification strategies yield the highest purity and activity for recombinant C. glabrata RPE1?

A multi-step purification strategy is recommended to obtain high-purity, active recombinant C. glabrata RPE1:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA or similar matrices for His-tagged proteins.

• Intermediate purification: Ion exchange chromatography (typically DEAE or Q Sepharose) to separate based on charge properties.

• Polishing step: Size-exclusion chromatography to isolate the correctly assembled hexameric form and remove aggregates or incomplete assemblies.

• Buffer optimization: Final preparation in 30 mM Tris-HCl (pH 7.9) or similar buffer systems that maintain stability and activity.

Throughout purification, it's critical to monitor enzyme activity using the coupled spectrophotometric assays. The active hexameric form typically elutes at volumes corresponding to approximately 300-400 kDa, which can be confirmed by analytical SEC and immunoblotting with anti-RPE1 antibodies .

What techniques are most effective for determining the quaternary structure of C. glabrata RPE1?

Multiple complementary techniques should be employed to accurately determine the quaternary structure of C. glabrata RPE1:

Comparison of recombinant protein with native enzyme extracted from C. glabrata cultures using immunoblotting can confirm that the observed quaternary structure exists in vivo rather than being an artifact of recombinant expression .

How can researchers accurately map the catalytic site and determine critical residues in C. glabrata RPE1?

To map the catalytic site and identify critical residues in C. glabrata RPE1, researchers should employ a combination of structural and functional approaches:

• Crystal structure analysis: Obtaining high-resolution crystal structures (preferably <2.0 Å) of RPE1 in complex with substrates, products, or inhibitors reveals the architecture of the active site. The catalytic pocket is typically located on top of the TIM-barrel fold .

• Site-directed mutagenesis: Systematically mutate predicted catalytic residues based on sequence alignments with characterized RPEs and assess the impact on enzyme activity.

• Enzyme kinetics with mutants: Determine changes in kinetic parameters (KM, kcat, catalytic efficiency) for mutations of key residues to quantify their contribution to catalysis.

• Inhibitor studies: Analyze the binding mode of competitive inhibitors to further characterize the active site geometry.

• Molecular dynamics simulations: Complement experimental data with computational analysis of substrate binding and catalytic mechanism.

By integrating these approaches, researchers can develop a comprehensive map of the catalytic machinery and reaction mechanism of C. glabrata RPE1, which may reveal unique features compared to homologs from other species .

What are the optimal assay conditions for measuring both forward and reverse reactions catalyzed by C. glabrata RPE1?

For accurate measurement of C. glabrata RPE1 activity in both reaction directions, the following optimized conditions are recommended:

For PPP-related activity (Ru5P → X5P):

• Buffer: 50 mM Tris-HCl (pH 7.9)

• Co-factors: 15 mM MgCl₂, 0.1 mM thiamine pyrophosphate (TPP)

• Additional components: 0.1% bovine serum albumin

• Coupled enzymes: Transketolase (1 μM), triose-phosphate isomerase (4 nM), α-glycerophosphate dehydrogenase (2 units mL⁻¹)

• Substrate range: 0.25-4 mM Ru5P

• Detection: Spectrophotometric monitoring of NADH oxidation at 340 nm

• Temperature: 25°C

For CBB-related activity (X5P → Ru5P):
Similar conditions with X5P as the substrate (concentration range 0-1.5 mM) and appropriate coupling enzymes to detect Ru5P formation .

For both assays, it's essential to:

• Perform control reactions without RPE1 to account for substrate contamination

• Ensure linear relationship between enzyme concentration and activity

• Pre-separate RPE1 by size-exclusion chromatography to ensure the hexameric form is being analyzed

• Include appropriate blanks to correct for background NADH oxidation

How do the kinetic parameters of C. glabrata RPE1 compare with those from other species, and what implications does this have for its biological function?

While specific kinetic data for C. glabrata RPE1 is not directly provided, comparative analysis with characterized RPEs from other organisms reveals important patterns:

*Expected ranges are extrapolated based on typical variations between species .

These patterns suggest that:

• RPE1 typically has higher affinity for X5P compared to Ru5P

• The catalytic efficiency for the Ru5P→X5P direction is generally greater than the reverse reaction

• The difference in directional efficiency likely reflects the metabolic role of RPE1 in the organism

For C. glabrata, a non-photosynthetic pathogenic yeast, the kinetic parameters would be expected to favor the PPP direction to support NADPH production and pentose generation, which are critical for survival during oxidative stress encountered within host environments .

What role do post-translational modifications play in regulating C. glabrata RPE1 activity?

Post-translational modifications likely play significant roles in regulating C. glabrata RPE1 activity, though specific modifications must be experimentally determined. Based on studies of RPE1 from other species, several regulatory mechanisms can be anticipated:

• Phosphorylation: In Chlamydomonas, RPE1 undergoes phosphorylation at multiple sites including Ser50, Thr220, and Ser239 . The location of these modifications at the entrance of the catalytic cleft suggests a phosphorylation-based regulatory mechanism. C. glabrata RPE1 likely contains analogous phosphorylation sites that modulate enzyme activity in response to metabolic demands.

• Redox regulation: Although CrRPE1 was found not to be regulated by thiol-switching mechanisms despite being identified as a putative target of thiol-based redox modifications , C. glabrata RPE1 may differ in this respect given the importance of redox balance in pathogenic fungi during host interaction.

• Other modifications: Additional modifications such as acetylation, methylation, or glycosylation might contribute to regulation of C. glabrata RPE1, requiring specific proteomic analyses to identify.

Understanding these regulatory mechanisms is particularly important in C. glabrata, as they may be linked to virulence and stress responses that enable this pathogen to survive within human hosts .

How can researchers distinguish between regulatory modifications and structural requirements in C. glabrata RPE1?

To differentiate between regulatory modifications and structural requirements in C. glabrata RPE1, researchers should implement a systematic approach:

• Comprehensive mapping of modifications: Use mass spectrometry-based proteomic approaches to identify all post-translational modifications (PTMs) present on natively expressed and recombinant RPE1.

• Structural mapping: Position identified modifications on the three-dimensional structure to determine if they are:

  • Located near the catalytic site (suggesting direct regulation)

  • Found at subunit interfaces (suggesting quaternary structure regulation)

  • Positioned at exposed surfaces (potentially involved in protein-protein interactions)

  • Located in core structural regions (suggesting structural roles)

• Site-directed mutagenesis: Create mutants that either:

  • Prevent modification (e.g., Ser→Ala to prevent phosphorylation)

  • Mimic constitutive modification (e.g., Ser→Asp to mimic phosphorylation)

• Functional assays: Assess how each mutation affects:

  • Enzyme activity and kinetic parameters

  • Oligomeric assembly

  • Protein stability

  • Response to cellular stressors

• In vivo validation: Complement C. glabrata RPE1 deletion strains with wild-type or mutant variants to assess physiological relevance of modifications during growth under various conditions, particularly those mimicking host environments .

By integrating these approaches, researchers can build a comprehensive model of how specific modifications contribute to either structural integrity or dynamic regulation of RPE1 activity in C. glabrata.

What evolutionary insights can be gained from comparing C. glabrata RPE1 with homologs from other fungi and non-fungal organisms?

Comparative analysis of C. glabrata RPE1 with homologs from diverse organisms offers valuable evolutionary insights:

• Functional conservation vs. specialization: While the core catalytic mechanism of RPE1 is likely conserved across species, C. glabrata RPE1 may exhibit adaptations related to its lifestyle as an opportunistic pathogen. Comparing with non-pathogenic yeasts and photosynthetic organisms like Chlamydomonas can reveal specializations related to pathogenicity .

• Structural variations: Although the basic TIM-barrel fold and hexameric assembly are likely conserved, subtle structural differences may exist in substrate binding pockets or oligomerization interfaces. For instance, while CrRPE1 forms a homo-hexamer, RPE1 from other species might exhibit different quaternary structures adapted to their specific metabolic contexts .

• Regulatory divergence: The mechanisms regulating RPE1 activity likely diverged during evolution. While the Chlamydomonas enzyme appears to be regulated primarily through phosphorylation rather than redox mechanisms , C. glabrata RPE1 may have evolved distinct regulatory mechanisms reflecting its adaptation to fluctuating environments encountered during infection.

• Subcellular localization: In photosynthetic organisms like Chlamydomonas, RPE isoforms show differential localization between chloroplast and cytosol . In C. glabrata, which lacks chloroplasts, understanding the subcellular distribution of RPE1 can provide insights into metabolic compartmentalization in pathogenic fungi.

What methods should be used to perform comprehensive sequence and structural comparison between RPE1 enzymes from different species?

For rigorous comparative analysis of RPE1 enzymes across species, researchers should employ a multi-layered approach:

• Sequence analysis pipeline:

  • Multiple sequence alignment using MUSCLE, MAFFT, or T-Coffee algorithms

  • Phylogenetic tree construction using maximum likelihood or Bayesian methods

  • Conservation analysis to identify universally conserved residues versus species-specific variations

  • Motif identification focusing on catalytic sites and regulatory regions

• Structural comparison techniques:

  • Superposition of available crystal structures using PyMOL or UCSF Chimera

  • Root-mean-square deviation (RMSD) calculation for backbone and side-chain atoms

  • Analysis of B-factors to identify regions of flexibility differences

  • Electrostatic surface potential comparison to detect species-specific charge distributions

  • Molecular dynamics simulations to compare dynamic behavior

• Functional correlation approaches:

  • Map sequence and structural differences to experimentally determined kinetic parameters

  • Correlate species-specific variations with ecological niches or metabolic strategies

  • Perform ancestral sequence reconstruction to trace evolutionary trajectories

• Experimental validation:

  • Create chimeric enzymes swapping domains between species

  • Introduce species-specific residues through site-directed mutagenesis

  • Express and characterize enzymes from diverse species under identical conditions

This integrated approach will illuminate how evolutionary pressures have shaped RPE1 structure and function across species, with particular focus on adaptations in C. glabrata related to its pathogenic lifestyle.

How does RPE1 activity contribute to C. glabrata virulence and survival within the host environment?

RPE1 likely plays crucial roles in C. glabrata virulence and host survival through several interconnected mechanisms:

• NADPH production for oxidative stress resistance: As a key enzyme in the PPP, RPE1 contributes to the generation of NADPH, which is essential for maintaining redox balance and detoxifying reactive oxygen species (ROS) produced by host immune cells. This function is particularly important for C. glabrata, which must contend with oxidative bursts from macrophages and neutrophils during infection .

• Metabolic flexibility: RPE1's role in carbohydrate metabolism enables C. glabrata to utilize diverse carbon sources within the nutrient-limited host environment. This metabolic adaptability allows the pathogen to thrive in different host niches where glucose availability may be restricted.

• Nucleotide biosynthesis support: By participating in the PPP, RPE1 contributes to the production of ribose-5-phosphate, a precursor for nucleotide synthesis. This supports the rapid growth and proliferation required during infection establishment .

• Biofilm formation: Robust carbohydrate metabolism, supported by enzymes like RPE1, is essential for producing extracellular matrix components needed for biofilm formation, which enhances C. glabrata resistance to antifungal treatments and host defenses .

• Age-related virulence: C. glabrata infections are more common in older adults, suggesting that RPE1's role in metabolism may intersect with age-related changes in host immunity or metabolism .

Understanding these connections provides potential targets for therapeutic intervention, as disrupting RPE1 function could simultaneously compromise multiple aspects of C. glabrata pathogenicity.

What experimental approaches can determine the impact of RPE1 deletion or inhibition on C. glabrata fitness and virulence?

To rigorously assess the impact of RPE1 manipulation on C. glabrata pathogenicity, researchers should employ a multi-faceted experimental approach:

• Genetic manipulation strategies:

  • CRISPR-Cas9 or traditional homologous recombination to create RPE1 deletion mutants

  • Conditional expression systems (tetracycline-regulated promoters) for controlled RPE1 depletion

  • Point mutations of catalytic residues to create enzymatically inactive variants

  • Complementation with wild-type RPE1 to confirm phenotype specificity

• In vitro phenotypic characterization:

  • Growth rate determination in various carbon sources

  • Stress resistance assays (oxidative, osmotic, pH, temperature)

  • Biofilm formation capacity

  • Antifungal susceptibility testing

  • Metabolomic profiling to measure PPP intermediates and products

• Host cell interaction studies:

  • Adhesion and invasion assays with epithelial cell lines

  • Survival within macrophages and neutrophils

  • Cytokine induction profiles

  • Host cell damage assays

• In vivo infection models:

  • Murine systemic candidiasis model

  • Tissue-specific infection models (vaginal, oral, gastrointestinal)

  • Competitive infection assays (wild-type vs. RPE1 mutant)

  • In vivo imaging to track fungal dissemination

• Inhibitor-based approaches:

  • Screen for selective RPE1 inhibitors

  • Evaluate inhibitor efficacy in vitro and in vivo

  • Assess synergy with existing antifungals

  • Determine inhibitor selectivity for fungal versus human RPE

These approaches would provide comprehensive insights into the contribution of RPE1 to C. glabrata fitness and virulence, potentially identifying new therapeutic strategies for difficult-to-treat C. glabrata infections, which are often resistant to azole antifungals .

How can structural information about C. glabrata RPE1 be leveraged for selective inhibitor design?

Structural insights into C. glabrata RPE1 provide valuable foundations for rational design of selective inhibitors through the following approaches:

• Active site targeting strategy:

  • Identify unique structural features of the C. glabrata RPE1 catalytic pocket compared to human homologs

  • Focus on differences in amino acid composition, pocket depth, hydrophobicity patterns, and charge distribution

  • Design transition state analogs that exploit fungal-specific interactions

  • Develop competitive inhibitors that mimic substrate binding but incorporate non-hydrolyzable modifications

• Allosteric inhibition approach:

  • Map allosteric sites unique to fungal RPE1 using molecular dynamics simulations

  • Target hexamer assembly interfaces to disrupt quaternary structure

  • Design inhibitors that lock the enzyme in an inactive conformation

  • Exploit species-specific regulatory sites identified through structural analysis

• Structure-based virtual screening workflow:

  • Develop high-quality homology models if crystal structures are unavailable

  • Perform molecular docking of large compound libraries against identified pockets

  • Prioritize compounds based on predicted binding energy and selectivity

  • Refine hits through fragment-based approaches and structure-activity relationship studies

• Experimental validation pipeline:

  • Enzymatic assays to confirm inhibitory activity

  • Thermal shift assays to verify target engagement

  • X-ray crystallography of enzyme-inhibitor complexes

  • Cellular activity assays to confirm antifungal properties

  • Selectivity profiling against human RPE homologs

This integrated approach maximizes the probability of developing potent and selective RPE1 inhibitors as potential antifungal agents against C. glabrata infections, which are often resistant to conventional treatments .

What are the technical challenges in expressing and characterizing mutant variants of C. glabrata RPE1, and how can they be overcome?

Expressing and characterizing mutant variants of C. glabrata RPE1 presents several technical challenges that require specific solutions:

By systematically addressing these challenges, researchers can generate reliable structure-function data on C. glabrata RPE1 mutants, advancing understanding of this enzyme's role in fungal metabolism and potential as a therapeutic target.