Recombinant Candida glabrata Ribose-5-phosphate isomerase (RKI1)

What is Ribose-5-phosphate isomerase (RKI1) and what is its function in Candida glabrata?

Ribose-5-phosphate isomerase (RKI1) in Candida glabrata is an enzyme with EC number 5.3.1.6 that catalyzes the interconversion of D-ribose-5-phosphate and D-ribulose-5-phosphate in the non-oxidative branch of the pentose phosphate pathway. The enzyme plays crucial roles in pentose-phosphate shunt and pyridoxine biosynthetic process, with localization in both the cytoplasm and nucleus . Its alternative names include D-ribose-5-phosphate ketol-isomerase and Phosphoriboisomerase .

How should recombinant RKI1 be stored for optimal stability and activity?

Recombinant RKI1 stability depends on multiple factors including buffer composition, storage temperature, and formulation. For optimal storage conditions:

Working aliquots can be stored at 4°C for up to one week, but repeated freezing and thawing should be avoided as it may compromise enzyme activity . For reconstitution, briefly centrifuge the vial prior to opening and reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL with 5-50% glycerol as a cryoprotectant (50% glycerol is recommended) .

How does RKI1 contribute to Candida glabrata metabolism and pathogenicity?

RKI1's role in the pentose phosphate pathway positions it as a critical enzyme for:

• NADPH generation (via the oxidative branch of PPP)

• Nucleotide biosynthesis (through ribose-5-phosphate production)

• Aromatic amino acid biosynthesis

• Oxidative stress response (by maintaining NADPH levels)

While direct evidence from the search results is limited, similar enzymes in related organisms like Trypanosoma brucei have been shown to be essential for parasite growth and infectivity . RKI1 may play comparable roles in Candida glabrata virulence by supporting metabolic adaptability during infection. Evidence for similar enzymes in T. brucei showed that knockdown resulted in reduced parasite growth in vitro and decreased infectivity in vivo .

What experimental approaches can be used to measure RKI1 enzymatic activity?

RKI1 activity can be assessed using several methodological approaches:

For the spectrophotometric approach, researchers should use coupling enzymes like transketolase and glyceraldehyde-3-phosphate dehydrogenase to link RKI1 activity to measurable NADH oxidation/reduction.

How does Candida glabrata RKI1 compare to similar enzymes in other organisms?

Ribose-5-phosphate isomerases are classified into two structurally unrelated types:

• Type A (RpiA): Found in humans and most organisms

• Type B (RpiB): Found in some bacteria and unicellular eukaryotes including trypanosomatids

Interestingly, humans possess only type A ribose-5-phosphate isomerase, while Candida glabrata and trypanosomatids have type B . This structural difference makes RKI1 potentially attractive as a drug target since selective inhibition might be possible without affecting the human enzyme. Sequence analysis and structural predictions would be needed to confirm the classification of C. glabrata RKI1 and identify unique features that could be exploited for selective targeting.

What expression systems are optimal for producing recombinant C. glabrata RKI1?

E. coli is the predominant expression system for recombinant C. glabrata RKI1 as evidenced by the CUSABIO product information . When designing expression strategies, researchers should consider:

For E. coli expression, BL21(DE3) or Rosetta strains are recommended for high yield. Optimal expression typically involves:

• Induction with 0.5-1.0 mM IPTG

• Expression at lower temperatures (16-25°C) to improve solubility

• Use of solubility-enhancing tags (e.g., MBP, SUMO, or TRX)

• Lysis in buffers containing 20-50 mM phosphate or Tris (pH 7.5-8.0), 150-300 mM NaCl, and potentially glycerol (5-10%)

What purification strategies yield the highest purity and activity for RKI1?

For efficient purification of recombinant RKI1:

• Initial capture:

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

  • Glutathione affinity for GST-tagged protein

  • Amylose resin for MBP-tagged protein

• Intermediate purification:

  • Ion exchange chromatography based on RKI1's pI

  • Hydrophobic interaction chromatography

• Polishing step:

  • Size exclusion chromatography to remove aggregates and obtain >95% purity

The commercial recombinant protein achieves >85% purity as assessed by SDS-PAGE . For optimal activity retention, all purification steps should be performed at 4°C with buffers containing stabilizing agents such as glycerol.

What factors influence RKI1 activity and how can they be optimized in research settings?

Several factors affect recombinant RKI1 activity:

Each of these parameters should be systematically optimized using appropriate activity assays to determine conditions yielding maximum enzyme activity and stability.

How might RKI1 serve as a potential antifungal target?

RKI1 represents a promising antifungal target for several reasons:

• Essential metabolic function in the pentose phosphate pathway

• Structural differences from the human isozyme (type B vs. type A)

• Potential role in stress response and virulence

Research on similar enzymes in Trypanosoma brucei has demonstrated that knockdown of ribose-5-phosphate isomerase B compromises parasite growth and infectivity . This suggests that targeting RKI1 in C. glabrata might similarly impair fungal survival and virulence.

A target validation approach should include:

• Gene deletion or knockdown studies to assess essentiality

• Chemical inhibition studies with candidate compounds

• In vitro and in vivo infection models to evaluate impact on virulence

• Structural analysis to identify unique binding sites absent in human RpiA

What is known about the relationship between RKI1 and biofilm formation in Candida glabrata?

While the search results don't provide direct evidence linking RKI1 specifically to biofilm formation, the thesis by Diana Pereira mentions RKI1 in the context of C. glabrata biofilm research . Biofilm formation in C. glabrata involves complex transcriptional regulation, with transcription factors like Tec1 playing significant roles.

To investigate potential relationships between RKI1 and biofilm formation, researchers could:

• Analyze RKI1 expression profiles during different stages of biofilm development

• Create RKI1 knockout or knockdown strains and assess their biofilm-forming capacity

• Evaluate metabolic changes in pentose phosphate pathway flux during biofilm formation

• Assess whether RKI1 is differentially regulated by biofilm-specific transcription factors like Tec1

These approaches could reveal whether RKI1 contributes to the metabolic adaptations required during biofilm formation.

What computational approaches can be used to identify potential RKI1 inhibitors?

Several computational strategies can accelerate the discovery of selective RKI1 inhibitors:

The lack of human RpiB makes this approach particularly promising for selective targeting. Researchers should focus on unique structural features of the C. glabrata enzyme that differ from human RpiA to maximize selectivity.

How can researchers study RKI1 function in the context of C. glabrata stress responses?

To investigate RKI1's role in stress responses:

• Expression analysis:

  • qRT-PCR to quantify RKI1 expression under various stressors (oxidative, osmotic, antifungal)

  • Western blotting to assess protein levels and potential post-translational modifications

• Genetic manipulation:

  • CRISPR-Cas9 or traditional gene disruption to create knockout strains

  • Conditional expression systems to modulate RKI1 levels

  • Site-directed mutagenesis to create activity-impaired variants

• Metabolomic analysis:

  • LC-MS to quantify pentose phosphate pathway metabolites under stress conditions

  • 13C-flux analysis to measure pathway activity in wild-type vs. RKI1-modified strains

• Phenotypic assays:

  • Growth inhibition assays under various stress conditions

  • ROS detection assays to measure oxidative stress resistance

  • Combined stress tests to assess adaptability

These methodologies would help elucidate RKI1's contribution to stress adaptation in C. glabrata, potentially revealing new therapeutic vulnerabilities.

What are the most pressing unanswered questions about C. glabrata RKI1?

Despite available information, several critical knowledge gaps remain:

• The precise three-dimensional structure of C. glabrata RKI1

• Detailed kinetic parameters and substrate specificity

• Regulation of RKI1 expression during infection and stress

• Direct evidence for RKI1's role in virulence and pathogenesis

• Potential for selective inhibition as an antifungal strategy

Addressing these questions will require integrated experimental approaches combining structural biology, biochemistry, molecular genetics, and infection models.

Future research priorities should include structural determination, development of selective inhibitors, and in vivo validation of RKI1 as a potential drug target, leveraging the apparent absence of RpiB in humans as a selectivity advantage .