Recombinant Oryza sativa subsp. japonica Inosine triphosphate pyrophosphatase (Os10g0457500, LOC_Os10g31940)

What is Inosine Triphosphate Pyrophosphatase (ITPase) and what is its primary function in rice?

Inosine triphosphate pyrophosphatase (ITPase) in rice (Oryza sativa subsp. japonica) is a critical enzyme encoded by the Os10g0457500 gene (LOC_Os10g31940). This enzyme functions as a pyrophosphatase that hydrolyzes non-canonical purine nucleotides such as inosine triphosphate (ITP), deoxyinosine triphosphate (dITP), or xanthosine 5'-triphosphate (XTP) to their respective monophosphate derivatives, with the simultaneous release of pyrophosphate .

The primary biological function of ITPase is to prevent the incorporation of these non-canonical purines into RNA and DNA, thereby maintaining genome integrity and avoiding chromosomal lesions . ITPase serves as a guardian of cellular nucleotide pools by ensuring that non-standard purine nucleotides are eliminated before they can be incorporated into nucleic acids during replication or transcription processes. The enzyme does not distinguish between the deoxy and ribose forms of these nucleotides, allowing it to protect both DNA and RNA integrity .

In rice, as in other organisms, ITPase plays a crucial role in nucleotide metabolism, particularly in handling products of purine deamination reactions that occur naturally within cellular compartments . This housekeeping function is essential for maintaining high-fidelity DNA replication and preventing mutagenesis that could compromise plant development and reproduction.

What are the molecular characteristics of recombinant Oryza sativa ITPase?

Recombinant Oryza sativa ITPase (Os10g0457500, LOC_Os10g31940) exhibits the following molecular characteristics:

• Complete amino acid sequence: MSGAAAAAAR ALPKAVTFVT GNAKKLEEVR AILGSSIPFQ SLKLDLPELQ GEPEDISKEK ARMAASQVNG PVLVEDTCLC FNALKGLPGP YIKWFLEKTG HEGLNNLLLA YEDKSAFAMC IFSLALGPGE EPMTFVGKTA GKIVPARGPA DFGWDPVFQP DGFDQTYAEM PKSVKNQISH RGKALALVKE HFAAANYKVQ NDGSA

• Sequence length: 205 amino acids (full-length protein)

• Molecular weight: Calculated molecular weight of 21,939 Da, with observed molecular weight of approximately 21 kDa on SDS-PAGE

• Enzyme classification: Member of the HAM1 NTPase family

• Enzymatic function: Hydrolyzes non-canonical purine nucleotides to their monophosphate derivatives and pyrophosphate

The enzyme can be expressed in various host systems including E. coli, yeast, baculovirus, or mammalian cells, with each system potentially affecting post-translational modifications and activity . When properly expressed and purified, recombinant rice ITPase retains the catalytic activity of the native enzyme, allowing for detailed biochemical and structural characterization.

Unlike many enzymes that require specific cofactors, rice ITPase activity primarily depends on proper protein folding and the availability of its non-canonical nucleotide substrates. The molecular characteristics of rice ITPase reflect its evolutionary conservation across species, highlighting its fundamental importance in nucleotide metabolism and genome protection.

What are the known substrates for rice ITPase and what is the reaction mechanism?

Rice ITPase exhibits specificity for a defined set of non-canonical purine nucleotides. Based on the available research, the known substrates include:

• Inosine triphosphate (ITP)

• Deoxyinosine triphosphate (dITP)

• Xanthosine 5'-triphosphate (XTP)

• Deoxy-xanthosine triphosphate (dXTP)

The enzyme shows limited or no activity toward standard nucleoside triphosphates or nucleoside diphosphates and monophosphates. According to research findings, "not much activity has been seen towards other nucleoside triphosphates, and in IDP or IMP it is completely absent" .

The primary reaction catalyzed by ITPase follows this mechanism:

• The enzyme binds the non-canonical purine nucleotide substrate

• It catalyzes the hydrolysis of the substrate between the α and β phosphates

• This results in the formation of the corresponding nucleoside monophosphate

• Simultaneously, pyrophosphate (PPi) is released

The reaction can be represented as:
$ITP + H₂O → IMP + PP_i$

Similar reactions occur with the other substrates:
$dITP + H₂O → dIMP + PP_i$
$XTP + H₂O → XMP + PP_i$
$dXTP + H₂O → dXMP + PP_i$

These substrates are primarily formed during normal cellular metabolism, particularly through deamination reactions of canonical purine nucleotides. For example, ATP can be deaminated to form ITP, and GTP can be deaminated to form XTP . By removing these non-canonical nucleotides, ITPase prevents their incorporation into nucleic acids, which would otherwise lead to mutations and genomic instability.

What experimental methods are optimal for measuring rice ITPase activity in vitro?

Several methodological approaches can be adapted to accurately measure rice ITPase activity in vitro, each with specific advantages depending on the research question:

Colorimetric Pyrophosphate Detection Assay

This two-step approach is based on the detection of released pyrophosphate:

• Incubate purified ITPase with substrate (ITP, dITP, or XTP)

• The released pyrophosphate (PPi) is hydrolyzed to inorganic phosphate (Pi) by inorganic pyrophosphatase

• Pi is detected colorimetrically using malachite green or similar reagents

Important considerations: Control for background phosphatase activity in plant extracts, which can be substantial. Also note that ITP is known to inhibit human inorganic pyrophosphatase, which may influence results .

HPLC-Based Substrate Depletion/Product Formation Assay

This approach directly monitors the conversion of substrate to product:

• Incubate rice ITPase with ITP or other substrate

• Terminate the reaction at defined time points

• Separate and quantify remaining substrate and formed product (IMP) by HPLC

• Calculate reaction rates based on substrate disappearance or product formation

This method offers high specificity but requires specialized chromatography equipment.

Coupled Enzyme Spectrophotometric Assay

A continuous assay system that can provide real-time kinetics:

• Link ITPase activity to NADH oxidation through coupling enzymes

• Measure decrease in NADH absorbance at 340 nm

• Calculate enzyme activity based on the rate of NADH consumption

Optimal Reaction Conditions

For accurate measurement of rice ITPase activity, consider these parameters:

When developing an assay for rice ITPase, it's essential to include proper controls, such as enzyme-free and substrate-free reactions, and to validate the linearity of the assay with respect to both enzyme concentration and time.

What alternative splicing patterns have been observed in the rice Os10g0457500 gene?

While specific alternative splicing data for the Os10g0457500 gene (encoding rice ITPase) is not comprehensively documented in the literature, valuable insights can be gained from large-scale rice transcriptome studies that reveal trends in rice gene splicing patterns.

Research on the rice transcriptome using PacBio long-read sequencing technology has revealed substantial alternative splicing across the rice genome. According to these studies:

• Predominant splicing patterns: "Intron retention was the prevalent alternative splicing event and exon skipping was the least observed" . This suggests that the Os10g0457500 gene would most likely exhibit intron retention as its primary alternative splicing mechanism if it undergoes alternative splicing.

• Novel splice junctions: "Of 73,659 splice junctions, 12,755 (17%) represented novel splice junctions with canonical and non-canonical intron boundaries" . This indicates that rice genes frequently utilize previously uncharacterized splice sites.

• Isoform diversity: In detailed studies of specific rice genes, substantial isoform diversity has been observed. For example, "19 starch synthesis-related genes, defining 276 spliced isoforms of which 94 splice variants were novel" . This suggests that regulatory genes like ITPase might also exhibit multiple isoforms.

To definitively characterize alternative splicing patterns in the Os10g0457500 gene, researchers should apply long-read sequencing approaches focused on this specific locus. The PacBio Iso-Seq method described in the literature is particularly suitable: "A cDNA library was prepared from RNA extracted from leaves, roots, seeds, inflorescences, and panicles of O. sativa ssp. japonica var Nipponbare and sequenced on a PacBio Sequel platform" .

Tissue-specific and developmental-stage-specific patterns of alternative splicing may exist for the ITPase gene, potentially resulting in protein variants with altered activities or subcellular localizations. Such variations could contribute to the fine-tuning of ITPase activity under different physiological conditions or developmental stages in rice.

How does rice ITPase contribute to protection against oxidative stress and DNA damage?

Rice ITPase plays a critical role in protecting against oxidative stress-induced DNA damage through several interconnected mechanisms:

Removal of Oxidatively Damaged Nucleotides

Under oxidative stress conditions, purine nucleotides in the cellular pool undergo increased rates of oxidation and deamination. Adenine can be deaminated to hypoxanthine (forming inosine nucleotides), while guanine can be deaminated to xanthine (forming xanthosine nucleotides) . ITPase hydrolyzes the resulting ITP and XTP before they can be incorporated into nucleic acids, effectively serving as a second line of defense against oxidative damage.

Prevention of Mutagenesis

Research has demonstrated that "ITPA knockdown results in elevated mutagenesis in response to HAP treatment" . This indicates that ITPase normally prevents mutagenesis by removing damaged nucleotides that would otherwise cause base mispairing during DNA replication. By maintaining clean nucleotide pools, ITPase ensures high-fidelity DNA replication even under oxidative stress conditions.

Reduction of DNA Breaks

Studies show that ITPase deficiency sensitizes cells to "HAP-induced DNA breaks and apoptosis" . This suggests that accumulation of non-canonical nucleotides either directly promotes DNA strand breaks or interferes with DNA repair mechanisms. Rice ITPase therefore protects genome integrity by preventing the sequence of events leading to DNA fragmentation.

Protection During Environmental Stress

Rice plants regularly encounter environmental stressors that increase oxidative damage, including:

• Drought conditions

• High light intensity and UV radiation

• Temperature extremes

• Pathogen attacks

All these stressors increase reactive oxygen species (ROS) production, which in turn elevates nucleotide damage rates. ITPase activity becomes particularly crucial during these stress conditions to prevent the accumulation and incorporation of damaged nucleotides.

Complementary Action with DNA Repair Systems

ITPase works in concert with DNA repair systems:

• ITPase prevents incorporation of damaged nucleotides

• If damaged nucleotides are incorporated, repair systems like base excision repair remove them

• This two-tier protection ensures genome stability even under severe oxidative stress

This protective role suggests that ITPase activity might be an important factor in rice varieties' resilience to environmental stresses, potentially contributing to the adaptive capacity of different cultivars to challenging growing conditions.

What are the optimal conditions for expressing recombinant rice ITPase in E. coli?

Successful expression of recombinant rice ITPase in E. coli requires careful optimization of multiple parameters to ensure high yield of active enzyme. Based on research with recombinant ITPases and standard protocols for plant protein expression, the following conditions are recommended:

Expression System Optimization

Media and Growth Conditions

For optimal expression, use one of these approaches:

• Standard approach:

  • LB media supplemented with 0.4% glucose to prevent leaky expression

  • Grow at 37°C until target OD600

  • Shift to 16-20°C prior to induction

  • Continue expression for 16-18 hours

• Enhanced approach:

  • Terrific Broth (TB) or auto-induction media

  • Supplement with 5-10% glycerol

  • Co-express molecular chaperones (GroEL/ES) to improve folding

  • Include 0.1-0.5 mM MgSO4 as cofactor

Cell Lysis and Initial Purification

• Harvest cells by centrifugation (5000g, 10 min, 4°C)

• Resuspend in lysis buffer containing:

  • 20 mM Tris-HCl, pH 8.0

  • 300 mM NaCl

  • 5% glycerol

  • 1 mM DTT

  • 1 mM PMSF

  • Lysozyme (1 mg/ml)

  • DNase I (5 μg/ml)

• Lyse cells using sonication or high-pressure homogenization

• Clear lysate by centrifugation (20,000g, 30 min, 4°C)

• Purify using Ni-NTA affinity chromatography

These optimized conditions should yield soluble, active recombinant rice ITPase suitable for biochemical and structural studies. Preliminary expression trials testing multiple conditions in parallel are recommended to determine the optimal parameters for a specific construct .

What purification strategies yield the highest activity for recombinant rice ITPase?

Purifying recombinant rice ITPase while preserving its enzymatic activity requires a careful, multi-step approach. The following comprehensive purification strategy is designed to maximize both yield and activity:

Initial Capture: Immobilized Metal Affinity Chromatography (IMAC)

For His-tagged rice ITPase:

• Equilibrate Ni-NTA or TALON resin with binding buffer (20 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 8.0)

• Apply cleared cell lysate

• Wash extensively with binding buffer

• Elute with imidazole gradient (50-250 mM)

• Analyze fractions by SDS-PAGE and activity assays

• Pool high-activity fractions

Intermediate Purification: Ion Exchange Chromatography

• Dialyze IMAC-purified protein against low-salt buffer (20 mM Tris-HCl, 50 mM NaCl, pH 8.0)

• Apply to appropriate ion exchange column (based on theoretical pI)

• Elute with linear NaCl gradient (50-500 mM)

• Test fractions for activity

• Pool peak activity fractions

Polishing Step: Size Exclusion Chromatography

• Concentrate protein using centrifugal concentrators (10 kDa MWCO)

• Apply to size exclusion column (Superdex 75 or 200)

• Elute with storage buffer containing:

  • 20 mM Tris-HCl, pH 8.0

  • 300 mM NaCl

  • 5% Trehalose (stabilizing agent)

  • 30% Glycerol

  • 0.05% Tween 80

  • 1 mM DTT

• Analyze fractions for purity and activity

• Pool and concentrate purified protein

Stability Enhancement Strategies

To maintain high enzymatic activity throughout purification and storage:

• Temperature control: Perform all purification steps at 4°C

• Reducing environment: Include 1-5 mM DTT or 0.5-2 mM TCEP to prevent oxidation

• Protease inhibition: Add protease inhibitor cocktail during initial steps

• Buffer optimization: Test different pH values (7.5-8.5) and salt concentrations

• Stabilizing additives: Consider adding glycerol (10-30%), trehalose (5%), or arginine (50-100 mM)

Storage Recommendations

Search result recommends: "Store at < -20°C, stable for 6 months. Please minimize freeze-thaw cycles."

For optimal preservation of activity:

• Aliquot into small volumes to avoid repeated freeze-thaw cycles

• Flash-freeze in liquid nitrogen before transferring to -80°C for long-term storage

• For working stocks, store at -20°C for up to 6 months

• For short-term use, store working aliquots at 4°C for up to one week

This comprehensive purification approach should yield highly pure and enzymatically active recombinant rice ITPase suitable for detailed biochemical characterization and structural studies.

How can one design experiments to assess ITPase function in rice under various stress conditions?

Designing comprehensive experiments to assess ITPase function under stress conditions requires a multi-faceted approach that integrates gene expression, protein analysis, and physiological assessments. The following experimental design provides a framework for investigating rice ITPase function under stress:

Transcriptional Response Analysis

Experimental design:

• Grow rice plants under controlled conditions

• Subject plants to various stresses:

  • Drought (water withholding)

  • Salt stress (NaCl treatment)

  • Heat stress (38-42°C)

  • Cold stress (4-10°C)

  • Oxidative stress (H₂O₂ or paraquat treatment)

  • Pathogen infection (bacterial or fungal pathogens)

• Collect leaf, root, and reproductive tissue samples at multiple time points (0, 3, 6, 12, 24, 48, 72 hours)

• Extract total RNA using a method optimized for rice tissues

Analysis methods:

• RT-qPCR targeting Os10g0457500 (ITPase gene)

• Include reference genes (e.g., actin, ubiquitin) for normalization

• Compare with known stress-responsive genes as positive controls

• Calculate fold-change in expression relative to unstressed controls

Protein-Level Analysis

Experimental approach:

• Generate custom antibodies against rice ITPase or create epitope-tagged transgenic lines

• Extract total protein from stressed and control plants

• Perform western blot analysis to quantify ITPase protein levels

• Assess potential post-translational modifications using:

  • Phospho-specific antibodies

  • 2D gel electrophoresis

  • Mass spectrometry-based proteomics

Subcellular localization studies:

• Create GFP-tagged ITPase constructs for transient expression

• Examine localization using confocal microscopy

• Determine if protein localization changes during stress response

Enzymatic Activity Measurements

Activity assay protocol:

• Extract total protein from stressed and control plants

• Measure ITPase activity using methods outlined in Question #5

• Calculate specific activity (nmol/min/mg protein)

• Compare activity changes with expression levels

• Test activity with different substrates (ITP, dITP, XTP)

Genetic Manipulation Approaches

Generation of modified rice lines:

• Create CRISPR/Cas9 knockout lines targeting Os10g0457500

• Develop RNAi lines for partial ITPase suppression

• Generate overexpression lines using constitutive promoters

• Create lines with stress-inducible ITPase expression

Stress response analysis:

• Subject modified lines to various stresses

• Compare phenotypic responses:

  • Growth parameters (height, biomass)

  • Yield components (grain number, weight)

  • Stress tolerance indicators (ROS levels, lipid peroxidation)

  • Photosynthetic efficiency

  • Survival rates

Nucleotide Pool Analysis

Analytical approach:

• Extract nucleotides from stressed and control plants

• Quantify canonical and non-canonical nucleotides using HPLC-MS

• Calculate ITP/ATP and XTP/GTP ratios

• Correlate nucleotide ratios with ITPase expression/activity

DNA/RNA Damage Assessment

Damage quantification:

• Measure 8-oxoguanine levels as indicator of oxidative DNA damage

• Assess mutation frequencies using reporter systems

• Quantify DNA strand breaks using comet assay

• Examine RNA quality using Bioanalyzer

This comprehensive experimental approach will provide a detailed understanding of how ITPase functions during stress responses in rice and its importance for stress adaptation. The integration of molecular, biochemical, and physiological analyses will reveal the multifaceted role of ITPase in maintaining nucleotide pool quality and genome integrity under challenging environmental conditions.