Recombinant Saccharomyces cerevisiae Isocitrate dehydrogenase [NADP] cytoplasmic (IDP2)

What is the genomic organization of Saccharomyces cerevisiae IDP2?

IDP2 (isocitrate dehydrogenase [NADP+]) is encoded by a protein-coding gene located on chromosome XII of Saccharomyces cerevisiae S288C. The gene has the Entrez Gene ID 850871 and is referenced by the mRNA sequence NM_001182061.1, which encodes the protein NP_013275.1. The genomic characterization of S. cerevisiae chromosome XII was first comprehensively described in a 1997 study published in Nature and further elucidated in subsequent genomic analyses . When designing experiments involving IDP2, researchers should consider its chromosomal context and potential regulatory elements that may affect expression patterns.

How does S. cerevisiae IDP2 compare to isocitrate dehydrogenases in other organisms?

IDP2 from S. cerevisiae (NP_013275.1) belongs to a conserved family of isocitrate dehydrogenases found across various taxa. Comparative analysis reveals homologs in diverse organisms including:

This evolutionary conservation suggests functional importance and provides opportunities for comparative studies of enzyme structure and function . When designing experiments with IDP2, researchers can leverage this conservation to inform mutagenesis studies or to predict structure-function relationships based on knowledge from better-characterized homologs.

What are the optimal experimental designs for studying IDP2 activity in vitro?

When designing experiments to study IDP2 activity in vitro, a true experimental research design with appropriate controls is essential. The following methodological approach is recommended:

• Variable identification:

  • Independent variables: substrate concentration (isocitrate), cofactor concentration (NADP+), pH, temperature, and relevant divalent cations

  • Dependent variable: enzyme activity (measured as rate of NADPH formation)

  • Control variables: buffer composition, ionic strength, enzyme concentration

• Experimental treatments:

  • Systematically vary one parameter while keeping others constant

  • Include appropriate enzyme-free controls and heat-inactivated enzyme controls

• Randomization:

  • Perform experiments in random order to minimize systematic errors

  • Use technical replicates (minimum of three) and biological replicates (different enzyme preparations)

For kinetic characterization, measurements should be made under initial velocity conditions (typically <10% substrate consumption) . Based on similar studies with archaeal IDH, expected parameters for S. cerevisiae IDP2 might include Km values in the micromolar range for isocitrate and millimolar range for NADP+ .

How should researchers design experiments to investigate the role of divalent cations in IDP2 activity?

To investigate the role of divalent cations in IDP2 activity, a factorial experimental design is recommended:

• Experimental approach:

  • Test multiple divalent cations (Mg2+, Mn2+, Ca2+, Zn2+) at various concentrations

  • Include negative controls with chelating agents (e.g., EDTA) to ensure metal-free conditions

  • Use a two-way ANOVA design to assess both cation type and concentration effects

• Measurement protocol:

  • Pre-incubate the enzyme with each cation before initiating the reaction

  • Monitor activity spectrophotometrically by measuring NADPH formation at 340 nm

  • Calculate relative activity compared to optimal conditions

• Data analysis:

  • Determine EC50 values for each cation

  • Construct Hill plots to assess cooperativity in cation binding

Based on studies with similar enzymes like the archaeal IDH from "Candidatus Micrarchaeum harzensis," IDP2 may show promiscuity regarding divalent cations as cofactors . Understanding these cation preferences is crucial for optimizing reaction conditions and inferring physiological relevance.

What are the optimal systems and conditions for recombinant expression of S. cerevisiae IDP2?

For recombinant expression of S. cerevisiae IDP2, several expression systems can be employed, with E. coli being the most commonly used:

• E. coli expression system:

  • Recommended strain: E. coli Rosetta pRARE (addresses codon bias issues)

  • Expression vector: pBAD202 or similar with a 6xHis-tag for purification

  • Induction conditions: Optimize temperature (16-30°C), inducer concentration, and duration

• Cloning strategy:

  • Amplify the IDP2 gene using PCR with primers containing appropriate restriction sites or overlaps for assembly

  • For optimal results, use isothermal in vitro ligation for assembly with linearized vector

  • Verification by sequencing is essential before expression

• Expression optimization:

  • Test various media (LB, TB, auto-induction)

  • Optimize induction parameters (OD600 at induction, inducer concentration, temperature)

  • Monitor expression by SDS-PAGE analysis of time-course samples

Similar approaches have been successful for expressing archaeal IDH, where the gene was PCR-amplified and cloned into pBAD202 with a 6xHis-tag, followed by transformation into E. coli Rosetta pRARE . This methodology can be adapted for S. cerevisiae IDP2, with appropriate modifications for codon optimization if necessary.

What purification strategies yield the highest purity and activity for recombinant IDP2?

A multi-step purification approach is recommended to obtain high-purity, active recombinant IDP2:

• Initial capture:

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

  • Recommended buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, with an imidazole gradient for elution

  • Screen imidazole concentrations (20-40 mM) in washing buffer to minimize contaminants

• Intermediate purification:

  • Ion exchange chromatography (IEX) based on IDP2's theoretical pI

  • Size exclusion chromatography (SEC) to remove aggregates and further purify the protein

• Quality control:

  • Assess purity by SDS-PAGE (aim for >95%)

  • Verify identity by western blot and/or mass spectrometry

  • Determine specific activity using standardized assay conditions

• Storage conditions:

  • Test stability in various buffers (e.g., phosphate, Tris) with different additives (e.g., glycerol, DTT)

  • Aliquot and store at -80°C to avoid freeze-thaw cycles

The purification strategy should be optimized to achieve electrophoretic homogeneity while maintaining enzyme activity . Monitoring specific activity throughout purification steps is essential to ensure that the purification process preserves enzyme functionality.

How should researchers determine the kinetic parameters of recombinant IDP2?

To determine the kinetic parameters of recombinant IDP2, a systematic approach using steady-state kinetics is recommended:

• Experimental setup:

  • Use a spectrophotometric assay monitoring NADPH formation at 340 nm

  • Maintain constant temperature (typically 25°C or 30°C) and optimal pH

  • Include appropriate blanks and controls for each measurement

• Substrate kinetics:

  • Vary isocitrate concentration (typically 0.1-10× expected Km) while keeping NADP+ constant at saturating levels

  • Plot initial velocities against substrate concentration

  • Fit data to appropriate models (Michaelis-Menten, Hill, etc.) using non-linear regression

• Cofactor kinetics:

  • Vary NADP+ concentration while keeping isocitrate constant at saturating levels

  • Determine Km and kcat for NADP+

  • Assess cofactor specificity by comparing activity with NADP+ versus NAD+

• Data analysis:

  • Calculate Km, Vmax, kcat, and kcat/Km values

  • Construct Lineweaver-Burk, Eadie-Hofstee, or Hanes-Woolf plots as secondary validations

Based on studies with similar enzymes, S. cerevisiae IDP2 might exhibit Km values in the range of 50-100 μM for isocitrate and 1-2 mM for NADP+, with kcat values around 40 s⁻¹ . These parameters provide insight into the enzyme's efficiency and substrate preference.

What approaches are effective for analyzing the pH dependence of IDP2 activity?

To analyze the pH dependence of IDP2 activity, the following methodological approach is recommended:

• Buffer selection:

  • Use a series of overlapping buffer systems to cover the pH range 5.0-10.0

  • Recommended buffers: MES (pH 5.5-6.5), MOPS (pH 6.5-7.5), HEPES (pH 7.0-8.0), Tris (pH 7.5-9.0), and CAPS (pH 9.0-10.0)

  • Maintain constant ionic strength across all buffers

• Experimental design:

  • Measure enzyme activity at each pH under standard conditions

  • Plot relative activity versus pH

  • Determine pH optimum and the pH range for >50% activity

• Advanced analysis:

  • Measure Km and kcat at various pH values to distinguish between effects on binding and catalysis

  • Plot log(kcat) and log(kcat/Km) versus pH to determine pKa values of catalytically important residues

• Interpretation:

  • Correlate pH effects with known structural features and catalytic mechanism

  • Compare with pH dependence of homologous enzymes

Based on studies with archaeal IDH, S. cerevisiae IDP2 might show optimal activity around pH 8.0, which is slightly alkaline . Understanding the pH dependence provides insights into the enzyme's physiological role and optimal conditions for in vitro applications.

How does the NADP+ binding pocket of IDP2 influence its cofactor specificity?

The NADP+ binding pocket structure is a critical determinant of IDP2's cofactor specificity. Analysis should be conducted as follows:

• Structural analysis:

  • Perform sequence alignment with well-characterized IDHs to identify conserved residues in the NADP+ binding pocket

  • Use homology modeling based on crystal structures of related enzymes if a structure for S. cerevisiae IDP2 is not available

  • Analyze hydrogen bonding patterns and electrostatic interactions with the 2'-phosphate of NADP+

• Experimental approach:

  • Design site-directed mutagenesis experiments targeting key residues in the binding pocket

  • Assess changes in Km for NADP+ and NAD+ following mutations

  • Determine the ratio of activity with NADP+ versus NAD+ to quantify specificity changes

• Key considerations:

  • Pay particular attention to residues interacting with the 2'-phosphate of NADP+

  • Look for proline residues that might affect secondary structure and binding pocket geometry

Based on studies with archaeal IDH, the presence of a proline residue in the NADP+ binding pocket might cause a decrease in hydrogen bonding of the cofactor and a distortion of local secondary structure, potentially explaining a lower affinity for NADP+ . This structure-function relationship is likely conserved in S. cerevisiae IDP2 and influences its kinetic properties.

What are the critical residues for IDP2 catalytic activity, and how can they be investigated?

To identify and investigate critical residues for IDP2 catalytic activity, a combined bioinformatic and experimental approach is recommended:

• Bioinformatic analysis:

  • Perform multiple sequence alignment of IDP2 with homologs from different species

  • Identify strictly conserved residues, especially near the active site

  • Use structural information from homologs to predict the catalytic mechanism

• Experimental strategies:

  • Conduct alanine scanning mutagenesis of conserved residues

  • Perform more targeted substitutions based on chemical properties (e.g., Asp→Asn to eliminate charge)

  • Analyze the effects of mutations on Km, kcat, and substrate specificity

• Advanced techniques:

  • Use pH-rate profiles of wild-type and mutant enzymes to identify residues involved in acid-base catalysis

  • Employ isotope effects to elucidate rate-limiting steps

  • Consider X-ray crystallography or cryo-EM to determine structure

• Data interpretation:

  • Correlate kinetic changes with structural perturbations

  • Propose refined catalytic mechanisms based on experimental findings

For NADP-dependent isocitrate dehydrogenases, conserved residues typically include those involved in metal binding, isocitrate coordination, and proton transfer during catalysis. The specific identification of these residues in S. cerevisiae IDP2 would provide valuable insights into its catalytic mechanism and potential for engineering.

How does IDP2 expression and activity change under different metabolic conditions in S. cerevisiae?

To investigate changes in IDP2 expression and activity under different metabolic conditions, a comprehensive experimental approach is needed:

• Expression analysis:

  • Culture S. cerevisiae under various conditions (fermentative vs. respiratory growth, different carbon sources, stress conditions)

  • Quantify IDP2 mRNA levels using RT-qPCR

  • Measure protein levels using western blot with specific antibodies

  • Consider reporter gene assays (e.g., IDP2 promoter-GFP fusion) for real-time monitoring

• Activity measurements:

  • Prepare cell extracts from cultures grown under different conditions

  • Measure IDP2 activity using standardized assay conditions

  • Correlate activity with expression levels to identify post-translational regulation

• Experimental design considerations:

  • Use appropriate control genes/proteins for normalization

  • Include time-course analyses to capture dynamic responses

  • Consider both acute and chronic adaptations to changed conditions

• Advanced approaches:

  • Utilize proteomics to identify post-translational modifications under different conditions

  • Apply metabolomics to correlate IDP2 activity with metabolite levels

  • Conduct flux analysis to determine the contribution of IDP2 to NADPH production

This systematic approach will help elucidate the physiological role of IDP2 in redox balance, especially during the transition from fermentative to respiratory metabolism when NADPH requirements may change.

What experimental approaches can distinguish the specific roles of IDP2 versus other isocitrate dehydrogenase isoforms?

S. cerevisiae contains multiple isocitrate dehydrogenase isoforms, and distinguishing their specific roles requires targeted experimental designs:

• Genetic approaches:

  • Generate single and multiple knockout strains (ΔIDP1, ΔIDP2, ΔIDH1/2, and combinations)

  • Perform complementation studies with plasmid-expressed isoforms

  • Use controlled promoters to manipulate expression levels of specific isoforms

• Biochemical discrimination:

  • Develop isoform-specific activity assays based on differences in kinetic parameters, pH optima, or inhibitor sensitivity

  • Use subcellular fractionation to separate compartment-specific isoforms (cytosolic vs. mitochondrial)

  • Employ isoform-specific antibodies for immunoprecipitation and activity assays

• Physiological characterization:

  • Compare growth phenotypes of knockout strains under various conditions

  • Analyze metabolite profiles in strains with altered isoform expression

  • Measure NADPH/NADP+ ratios in different cellular compartments

• Experimental design considerations:

  • Control for compensatory mechanisms in knockout strains

  • Use appropriate controls to validate subcellular fractionation

  • Apply statistical techniques like factorial ANOVA to analyze complex datasets

This multifaceted approach will help elucidate the specific contribution of IDP2 to cellular NADPH production and redox homeostasis, distinguishing it from the roles of other isocitrate dehydrogenase isoforms.

How can researchers address contradictory findings in IDP2 research through robust experimental design?

When faced with contradictory findings in IDP2 research, researchers should implement the following methodological approach:

• Systematic review and meta-analysis:

  • Conduct a comprehensive literature review to identify all relevant studies

  • Extract data using a double extraction method to minimize errors (21.7% fewer errors compared to single extraction)

  • Perform a meta-analysis to quantitatively synthesize available evidence

• Experimental replication and extension:

  • Replicate key experiments using identical conditions to original studies

  • Systematically vary experimental parameters to identify sources of variability

  • Include positive and negative controls, as well as reference standards

• Controlling for confounding variables:

  • Identify potential confounding variables in previous studies (e.g., strain background, media composition, purification method)

  • Design factorial experiments to test the influence of these variables

  • Use randomization and blinding when feasible to minimize bias

• Statistical considerations:

  • Perform power analysis to ensure adequate sample size

  • Pre-register experimental designs and analysis plans

  • Use appropriate statistical tests and consider multiple testing corrections

What are the cutting-edge approaches for studying the real-time dynamics of IDP2 activity in living cells?

To study real-time dynamics of IDP2 activity in living cells, several cutting-edge approaches can be employed:

• Genetically encoded biosensors:

  • Develop FRET-based sensors for NADPH/NADP+ ratio

  • Create biosensors that respond to IDP2 activity or its products

  • Use these sensors for spatiotemporal monitoring of IDP2 function

• Live-cell imaging techniques:

  • Apply fluorescence lifetime imaging microscopy (FLIM) to measure changes in NADPH levels

  • Use single-molecule tracking to monitor IDP2 localization and mobility

  • Implement super-resolution microscopy to visualize IDP2 interactions with other proteins

• Real-time enzymatic assays:

  • Develop cell-permeable fluorescent substrates or products for IDP2

  • Use microfluidics to monitor enzyme activity under changing conditions

  • Apply isotope tracing with rapid sampling for metabolic flux analysis

• Experimental design considerations:

  • Include appropriate controls for autofluorescence and photobleaching

  • Validate biosensor responses with biochemical assays

  • Use mathematical modeling to interpret complex dynamics

These advanced approaches provide unprecedented insights into the dynamic regulation of IDP2 activity and its contribution to cellular metabolism, moving beyond traditional biochemical assays to understand function in the native cellular context.

What are the most promising directions for future research on S. cerevisiae IDP2?

Future research on S. cerevisiae IDP2 should focus on several promising directions:

• Systems biology integration:

  • Investigate IDP2's role in genome-scale metabolic models

  • Study its interactions within the broader NADPH-producing network

  • Apply multi-omics approaches to understand its regulation in the context of global cellular responses

• Structural biology:

  • Determine high-resolution structures of S. cerevisiae IDP2 using X-ray crystallography or cryo-EM

  • Conduct dynamic studies using HDX-MS or NMR to understand conformational changes during catalysis

  • Perform computational simulations to elucidate substrate binding and catalytic mechanism

• Biotechnological applications:

  • Explore IDP2 engineering for enhanced NADPH regeneration in biocatalysis

  • Investigate its potential role in metabolic engineering for the production of value-added compounds

  • Develop IDP2 variants with altered cofactor specificity or improved stability

• Evolutionary and comparative studies:

  • Analyze the evolution of IDP2 across fungal species and beyond

  • Investigate adaptations in IDP2 properties related to ecological niches

  • Compare regulatory mechanisms across species to identify conserved and divergent features

These research directions will not only advance our fundamental understanding of IDP2 but also potentially lead to biotechnological applications in metabolic engineering and biocatalysis.

How can researchers design a comprehensive experimental plan to fully characterize novel IDP2 mutations?

To fully characterize novel IDP2 mutations, a comprehensive experimental plan should include:

• Initial characterization:

  • Express and purify wild-type and mutant proteins using identical protocols

  • Perform basic enzymatic assays to determine Km, kcat, and substrate specificity

  • Assess pH and temperature optima and stability profiles

• Structural analysis:

  • Conduct circular dichroism (CD) spectroscopy to assess secondary structure changes

  • Perform thermal shift assays to evaluate stability differences

  • If possible, determine crystal structures or use homology modeling

• In vivo characterization:

  • Complement IDP2 deletion strain with mutant variants

  • Assess growth phenotypes under various conditions

  • Measure intracellular NADPH/NADP+ ratios and related metabolites

• Experimental design considerations:

  • Include multiple biological replicates (minimum of three)

  • Use factorial experimental designs to test interactions between mutations and environmental conditions

  • Apply appropriate statistical analysis to identify significant differences

• Advanced characterization:

  • Conduct pre-steady-state kinetics to identify rate-limiting steps

  • Perform isotope effect studies to elucidate reaction mechanism changes

  • Use molecular dynamics simulations to understand structural perturbations