Recombinant Actinidia deliciosa Delta-1-pyrroline-5-carboxylate synthase, partial

What is the structural organization of Actinidia deliciosa P5CS and how does it relate to function?

P5CS in kiwifruit, like in other eukaryotes, is a bifunctional enzyme that possesses two distinct catalytic domains: a γ-glutamyl kinase (γ-GK) domain and a γ-glutamyl phosphate reductase (γ-GPR) domain. The γ-GK domain phosphorylates glutamate to form γ-glutamyl phosphate, while the γ-GPR domain then converts this intermediate to γ-glutamic semialdehyde (GSA), which spontaneously cyclizes to P5C .

The enzyme typically contains an ATP-binding motif in the γ-GK domain and an NAD(P)H binding motif in the γ-GPR domain. Based on homology with other plant P5CS enzymes, the C-terminal half shows approximately 48% sequence identity with yeast γ-GPR and possesses an eleven-codon NAD(P)H binding motif . This bifunctional organization enables coordinated catalysis of sequential reactions in proline biosynthesis.

How has P5CS evolved in plants, and what evolutionary patterns are observed in Actinidia species?

Molecular phylogenetic analyses indicate that P5CS duplication events have occurred several times following the emergence of flowering plants and at different frequencies throughout the evolution of monocots and dicots . In most higher plants, including Actinidia species, two isoforms of P5CS have been identified: one constitutively expressed to satisfy proline demand for protein synthesis, and the other stress-induced .

The evolution of P5CS genes appears to have involved gene duplication followed by subfunctionalization, resulting in isoforms with distinct regulatory properties. Comparative analysis of Actinidia P5CS with those from other plant species can reveal conserved domains and lineage-specific adaptations that may relate to the specific environmental pressures faced by kiwifruit plants .

What are the key functional differences between the different isoforms of P5CS in plants?

In plants, including Actinidia species, P5CS typically exists in two main isoforms with distinct functional characteristics:

• Constitutive isoform (P5CS2-like):

  • Expressed ubiquitously across tissues

  • Primarily responsible for basal proline production for protein synthesis

  • May be more sensitive to feedback inhibition by ornithine

  • Essential for normal growth and development

• Stress-induced isoform (P5CS1-like):

  • Upregulated under stress conditions

  • Less sensitive to feedback inhibition

  • Contains stress-responsive elements in promoter regions

  • Critical for proline accumulation during stress response

In humans, two P5CS transcript variants generated by exon sliding encode protein isoforms differing by a two amino acid insert at the N-terminus of the γ-glutamyl kinase active site. The short form (P5CS.short) is highly expressed in the gut and is inhibited by ornithine, while the long form (P5CS.long) is expressed ubiquitously and is insensitive to ornithine . Similar alternative splicing mechanisms might exist in kiwifruit P5CS, though specific data on Actinidia deliciosa isoforms is limited.

What are optimal conditions for expressing recombinant A. deliciosa P5CS in prokaryotic systems?

Based on successful expression protocols for recombinant proteins from Actinidia species, the following conditions are recommended for P5CS expression in E. coli:

• Expression system:

  • E. coli BL21(DE3) or Rosetta strains to address potential codon bias

  • pET or pQE vectors with inducible promoters

• Expression conditions:

  • Induction at OD600 of 0.6-0.8

  • IPTG concentration: 0.1-0.5 mM

  • Temperature: 16-20°C (lower temperatures enhance solubility)

  • Duration: 16-20 hours

• Protein solubility enhancements:

  • Addition of 5-10% glycerol to growth media

  • Inclusion of 0.1-0.5% Triton X-100 in lysis buffer

  • Co-expression with chaperones if solubility issues arise

The recombinant protein should include an N-terminal histidine tag for efficient purification by immobilized metal ion affinity chromatography (IMAC), as demonstrated for other Actinidia proteins .

What purification strategies yield the highest activity for recombinant kiwifruit P5CS?

Purification of recombinant A. deliciosa P5CS with high enzymatic activity requires careful consideration of the bifunctional nature of the enzyme. Based on successful purification of other plant P5CS proteins and recombinant kiwifruit proteins:

• Initial purification steps:

  • IMAC using Ni-NTA resin for His-tagged protein

  • Gradient elution with 20-250 mM imidazole

  • Include 10% glycerol and 1-5 mM DTT in all buffers

• Secondary purification:

  • Ion exchange chromatography (typically Q-Sepharose)

  • Size exclusion chromatography to remove aggregates

  • Consider affinity chromatography with substrate analogs

• Critical considerations:

  • Maintain reducing conditions throughout (1-5 mM DTT or β-mercaptoethanol)

  • Include protease inhibitors in all buffers

  • Keep samples at 4°C during purification

  • Avoid freeze-thaw cycles; store aliquots at -80°C with 20% glycerol

Protein purity should be assessed by SDS-PAGE, with expected molecular weight around 75-80 kDa based on other plant P5CS proteins . Activity should be measured immediately after purification to ensure enzyme functionality.

How can researchers troubleshoot low expression yields or poor enzyme activity?

When encountering problems with recombinant kiwifruit P5CS expression or activity, systematic troubleshooting is essential:

• Low expression yields:

  • Optimize codon usage for E. coli

  • Test different expression vectors and promoter strengths

  • Examine mRNA stability and protein degradation

  • Use fusion partners that enhance solubility (SUMO, MBP)

• Poor enzyme activity:

  • Verify correct protein folding by circular dichroism

  • Ensure both catalytic domains are intact via limited proteolysis

  • Check for proper cofactor availability (ATP, Mg²⁺, NAD(P)H)

  • Test activity at different pH values (typically pH 7.0-8.5)

  • Examine potential inhibition by purification components

• Protein instability:

  • Monitor protein degradation during storage

  • Test stabilizing additives (glycerol, trehalose, specialized buffers)

  • Consider alternative tag positions if N-terminal tag affects activity

  • Perform thermal shift assays to identify stabilizing conditions

If activity remains problematic, expression in eukaryotic systems such as yeast or insect cells may provide better results for this complex bifunctional enzyme .

What are the most reliable methods for measuring P5CS activity in vitro?

For reliable measurement of A. deliciosa P5CS enzymatic activity, researchers should consider the following methodologies:

• Coupled spectrophotometric assay:

  • Monitor NAD(P)H oxidation at 340 nm (ε₃₄₀ = 6.22 mM⁻¹cm⁻¹)

  • Reaction mixture: 0.1 M Tris-HCl (pH 7.5), 10 mM glutamate, 5 mM ATP,
    0.2 mM NAD(P)H, 10 mM MgCl₂, 1 mM EDTA, enzyme

  • Include 0.01% Brij-35 detergent to stabilize activity

  • Follow linear decrease in absorbance over time

• Separate domain activity measurements:

  • γ-GK activity: Quantify ADP formation using coupled enzyme assays
    (pyruvate kinase and lactate dehydrogenase)

  • γ-GPR activity: Monitor NAD(P)H oxidation with γ-glutamyl phosphate as substrate

• Direct P5C measurement:

  • React with ninhydrin or o-aminobenzaldehyde to form colored complexes

  • Measure absorbance at appropriate wavelengths

  • Use calibration curves with P5C standards

When reporting activity, use the physiologically relevant concentrations of substrates (glutamate ~10 mM, NAD(P)H ~40 μM) to better reflect in vivo conditions .

How can researchers distinguish between the two functional domains of P5CS in activity assays?

To differentiate between the γ-GK and γ-GPR domain activities of kiwifruit P5CS:

• Sequential assay approach:

  • Measure γ-GK activity by quantifying ATP consumption or ADP formation
    using coupling enzymes (pyruvate kinase and lactate dehydrogenase)

  • Measure γ-GPR activity by providing γ-glutamyl phosphate directly and
    monitoring NAD(P)H oxidation

• Specific inhibitor approach:

  • Use proline to selectively inhibit the γ-GK domain

  • Use metal chelators to differentially affect the two domains

  • Apply specific site-directed mutations to inactivate one domain while
    preserving the other

• Domain-specific substrate analogs:

  • Test glutamate analogs that affect only the γ-GK domain

  • Use γ-glutamyl phosphate analogs specific to the γ-GPR domain

Table 1: Comparative kinetic parameters for different domain assays

What factors influence P5CS activity measurements and how can they be controlled?

Multiple factors can affect P5CS activity measurements, introducing variability or artifacts:

• Enzyme stability factors:

  • Temperature (maintain at 4°C until assay)

  • Oxidation of sulfhydryl groups (add 1-5 mM DTT or β-mercaptoethanol)

  • Proteolytic degradation (add protease inhibitors)

  • Protein concentration (dilute in buffer with 1 mg/ml BSA)

• Assay condition variables:

  • pH (optimal range typically pH 7.0-8.0)

  • Ionic strength (100-150 mM salt concentration optimal)

  • Divalent cations (Mg²⁺ concentration critical, typically 5-10 mM)

  • Temperature (standard assays at 25-37°C)

• Substrate and product effects:

  • Substrate quality (prepare fresh solutions)

  • Product inhibition (optimize reaction time to avoid accumulation)

  • NAD(P)H oxidation by contaminants (include appropriate blanks)

  • P5C/GSA non-enzymatic cyclization (account for spontaneous reaction)

To ensure reproducible results, standardize protein quantification methods, perform assays in technical triplicates, include appropriate controls, and use internal standards when possible .

How do mutations in key residues affect the catalytic activity of kiwifruit P5CS?

Mutations in conserved residues can profoundly impact P5CS catalytic activity, as demonstrated by studies of similar enzymes:

• ATP-binding domain mutations:

  • Alterations in the aspartokinase-like active site region can impair γ-GK activity

  • Mutations in the ATP-binding motif can reduce catalytic efficiency by 100-fold or more

  • Conserved lysine or arginine residue substitutions typically abolish γ-GK activity

• NAD(P)H-binding site mutations:

  • Modifications to the eleven-codon NAD(P)H binding motif affect cofactor binding

  • As demonstrated in PYCR1 studies, even single amino acid changes (e.g., T171M) can reduce kcat by 100-200-fold when P5C is the variable substrate

  • Alterations in the binding pocket can change cofactor preference (NADH vs. NADPH)

• Substrate binding region:

  • Mutations that alter substrate access (as shown in the T171M PYCR1 variant) can impair activity through steric hindrance

  • Changes in the active site can affect substrate specificity

  • Interdomain communication can be disrupted by mutations in linker regions

For structure-function studies of A. deliciosa P5CS, site-directed mutagenesis of conserved residues identified through sequence alignment with well-characterized P5CS enzymes would be most informative .

What structural features of P5CS are responsible for its regulatory properties?

The regulatory properties of P5CS are governed by several structural features:

Understanding these structural features in A. deliciosa P5CS would require detailed structural studies, potentially using homology modeling based on available templates (such as those listed in SWISS-MODEL with QMEAN scores of 0.77 and 0.76) .

How does the three-dimensional structure of A. deliciosa P5CS compare with those from other species?

While the specific three-dimensional structure of A. deliciosa P5CS has not been experimentally determined, comparative analysis can provide insights:

• Homology-based predictions:

  • SWISS-MODEL Repository identifies two potential oligomeric states for A. deliciosa P5CS: homo-2-mer (template 2h5g.1.B) and homo-8-mer (template 8y2h.1.A)

  • These templates show high QMEAN scores (0.77 and 0.76), suggesting reliable structural predictions

  • The homo-8-mer template includes bound ATP, indicating potential ligand binding sites

• Expected conserved features:

  • The γ-GK domain likely adopts an α/β fold similar to other kinases

  • The γ-GPR domain would contain a Rossmann fold for NAD(P)H binding

  • The interdomain linker region may be flexible to allow coordinated catalysis

• Species-specific variations:

  • Surface-exposed regions may show greater variability between species

  • Regulatory regions might differ between plant and animal P5CS enzymes

  • Substrate binding pockets could be adapted to specific metabolic contexts

To fully characterize the structure, experimental approaches such as X-ray crystallography or cryo-electron microscopy would be necessary, potentially using the purification methods outlined for recombinant expression of the protein .

How is P5CS expression regulated in A. deliciosa under different environmental conditions?

P5CS expression in Actinidia and other plants is regulated by multiple mechanisms in response to environmental conditions:

• Transcriptional regulation:

  • Stress conditions (drought, salinity) upregulate the stress-responsive isoform

  • Hormones can modulate expression (downregulation by hydrocortisone and dexamethasone, upregulation by estradiol in human studies)

  • In humans, p53 can upregulate P5CS.long during p53-induced apoptosis

• Epigenetic regulation:

  • DNA methylation may play a role in controlling P5CS expression

  • In 'Kohi' kiwifruit, the upstream region of certain genes shows higher methylation of cytosine residues compared to 'Hayward' cultivar

  • This epigenetic modification could potentially regulate P5CS expression in different kiwifruit varieties

• Post-translational regulation:

  • Feedback inhibition by proline

  • Potentially regulated by phosphorylation or other post-translational modifications

  • Controlled by changes in subcellular localization

Studies in kiwifruit specifically should examine transcript levels under various stress conditions using RT-qPCR, analyze promoter methylation patterns, and investigate hormone responses to fully characterize regulation .

What role does P5CS play in stress response mechanisms in kiwifruit?

P5CS plays crucial roles in stress response mechanisms in plants, likely including kiwifruit:

The dual role of P5CS in both normal metabolism and stress response highlights its importance in plant adaptation mechanisms. In kiwifruit specifically, understanding P5CS regulation could provide insights into stress tolerance mechanisms relevant for cultivation in changing climatic conditions .

How does P5CS activity influence other metabolic pathways in kiwifruit?

P5CS activity intersects with multiple metabolic pathways in plants:

These metabolic interconnections highlight the importance of P5CS beyond just proline biosynthesis, suggesting its potential role in broader aspects of kiwifruit physiology and fruit quality .

How can recombinant A. deliciosa P5CS be used to engineer stress tolerance in plants?

Recombinant A. deliciosa P5CS could be utilized for improving stress tolerance in plants through several approaches:

• Transgenic overexpression strategies:

  • Constitutive overexpression using strong promoters (e.g., CaMV 35S)

  • Stress-inducible expression using drought/salt-responsive promoters

  • Tissue-specific expression targeting key tissues

• Structure-informed protein engineering:

  • Modification of feedback inhibition sites to reduce proline-mediated inhibition

  • Engineering enhanced catalytic efficiency

  • Altering cofactor preference to optimize activity under stress conditions

• P5CS gene editing in crops:

  • CRISPR/Cas9-mediated modification of endogenous P5CS genes

  • Promoter engineering to enhance stress responsiveness

  • Precise modification of regulatory regions based on kiwifruit P5CS structure

The effectiveness of these approaches should be evaluated through comprehensive phenotypic analysis, including measurements of proline accumulation, stress tolerance parameters, and potential effects on growth and development .

What potential biotechnological applications exist for recombinant kiwifruit P5CS beyond stress tolerance?

Recombinant A. deliciosa P5CS has potential applications beyond improving stress tolerance:

• Metabolic engineering of specialized metabolites:

  • Modulation of proline levels might influence pathways leading to flavor compounds

  • Could be used to enhance fruit quality characteristics

  • Potential application in modifying kiwifruit flavor profiles

• Protein production systems:

  • Enhancing proline biosynthesis could support increased protein production

  • Applications in pharmaceutical protein expression systems

  • Improving recombinant protein folding and stability

• Biomedical applications:

  • Studies of P5CS deficiency models for human metabolic disorders

  • Investigation of P5CS as a potential drug target

  • Understanding the role of P5CS in cancer, given the upregulation of P5CS.long by p53 in cancer cells

• Enzyme technology:

  • Development of P5CS-based biosensors for glutamate or proline

  • Biocatalytic applications for specialty chemical synthesis

  • Immobilized enzyme systems for biotechnological processes

These diverse applications highlight the potential value of recombinant kiwifruit P5CS beyond agricultural applications .

What methodological approaches are most effective for studying the impact of modified P5CS expression in transgenic plants?

To effectively study the impact of modified P5CS expression in transgenic plants:

• Molecular characterization:

  • Quantify transgene expression using RT-qPCR

  • Verify protein levels via western blotting

  • Confirm enzyme activity using in vitro assays

  • Determine transgene copy number and insertion sites

• Metabolite analysis:

  • Measure proline levels using ninhydrin-based assays or HPLC

  • Analyze amino acid profiles to detect metabolic shifts

  • Perform untargeted metabolomics to identify broader metabolic changes

  • Use isotope labeling to track metabolic fluxes from glutamate to proline

• Stress response phenotyping:

  • Assess osmotic stress tolerance through controlled drought experiments

  • Measure salt tolerance using defined salt stress treatments

  • Quantify oxidative stress markers (MDA, H₂O₂, antioxidant enzymes)

  • Evaluate physiological parameters (photosynthesis, stomatal conductance)

• Field performance evaluation:

  • Compare growth and yield under normal and stress conditions

  • Assess fruit quality characteristics

  • Evaluate stress recovery capacity

  • Conduct multi-location trials under different environmental conditions

These comprehensive approaches provide a robust framework for assessing the effects of P5CS modification in transgenic plants .

How does the kinetic profile of A. deliciosa P5CS compare with P5CS enzymes from other plant species?

While specific kinetic data for A. deliciosa P5CS is not available in the search results, comparative analysis with other plant P5CS enzymes would examine:

A comprehensive kinetic characterization of recombinant A. deliciosa P5CS would enable meaningful comparisons with other plant P5CS enzymes and potentially reveal adaptations specific to kiwifruit metabolism .

What structural and functional variations exist among P5CS enzymes from different kiwifruit varieties?

Variations in P5CS structure and function among different kiwifruit varieties could include:

• Sequence polymorphisms:

  • Single nucleotide polymorphisms affecting catalytic or regulatory domains

  • Insertions/deletions in non-critical regions

  • Variations in transit peptides affecting subcellular localization

• Promoter variations:

  • Differences in DNA methylation patterns, as observed between 'Kohi' and 'Hayward' cultivars for other genes

  • Polymorphisms in stress-responsive elements

  • Variations affecting hormone-responsive elements

• Expression patterns:

  • Cultivar-specific expression levels under normal and stress conditions

  • Differential tissue-specific expression

  • Varied stress-responsiveness of different isoforms

• Alternative splicing:

  • Potential differences in splicing patterns similar to human P5CS.short and P5CS.long

  • Variety-specific exon usage

  • Stress-induced alternative splicing

Comparative analysis across varieties like 'Hayward', 'Hort16A', and 'Kohi' could reveal adaptations related to different environmental niches or fruit quality characteristics .

How do photoselective nets influence P5CS expression and proline metabolism in kiwifruit cultivation?

Based on related studies in kiwifruit cultivation, photoselective nets (PNs) could influence P5CS expression and proline metabolism:

• Light quality effects:

  • Different PNs (yellow, pearl, gray) with varying shading degrees (4%, 7%, 19%) alter the light spectrum

  • Changed light quality could affect transcription factors regulating P5CS expression

  • Potential impact on photosynthesis-derived metabolites that feed into proline biosynthesis

• Physiological responses:

  • PNs influence several physiological traits in kiwifruit, including pollen traits

  • Similar effects might extend to stress response mechanisms

  • Altered microclimate under PNs could affect osmotic status and proline requirements

• Experimental approaches to investigate PN effects:

  • Compare P5CS transcript levels in tissues from plants grown under different PNs

  • Measure proline content in leaves and fruits under various PN conditions

  • Analyze stress marker expression in conjunction with P5CS expression

  • Evaluate fruit quality parameters in relation to proline content

This research direction could provide valuable insights for optimizing cultivation practices to enhance stress tolerance while maintaining fruit quality .

How does the mitochondrial localization of P5CS influence its role in cellular metabolism in kiwifruit?

The mitochondrial localization of P5CS has significant implications for its metabolic functions:

Advanced research should investigate the submitochondrial localization of kiwifruit P5CS, its interaction with the electron transport chain, and the mechanisms managing potentially toxic P5C levels to prevent detrimental effects on mitochondrial function .

What role does P5CS play in the regulation of flowering time and reproductive development in kiwifruit?

Evidence from Arabidopsis suggests P5CS may influence flowering and reproductive development:

• Flowering time regulation:

  • Arabidopsis p5cs1 mutants exhibit delayed floral transition

  • Transgenic Arabidopsis overexpressing P5CS1 show striking anticipation of flowering time

  • Proline may function as a floral signal interacting with flower regulators

• Coflorescence architecture:

  • P5CS1 overexpression in Arabidopsis led to proliferation of coflorescences

  • This effect was particularly pronounced under short-day conditions

  • Suggests role in regulating meristem identity and function

• Potential mechanisms in kiwifruit:

  • P5CS might influence floral transition through interaction with flowering regulators like CONSTANS (CO) and FLOWERING LOCUS C (FLC)

  • Proline's role as a signaling molecule rather than osmolyte in this context

  • Possible connections to photoperiodic, autonomous, vernalization or gibberellin pathways

Investigation of P5CS expression during kiwifruit floral development and analysis of proline content in meristems during the vegetative-to-reproductive transition could provide insights into similar roles in Actinidia species .

How do epigenetic mechanisms regulate P5CS expression in different kiwifruit cultivars?

Epigenetic regulation of P5CS may contribute to cultivar-specific traits in kiwifruit:

• DNA methylation patterns:

  • Different kiwifruit cultivars show varying DNA methylation patterns

  • The 'Kohi' cultivar exhibits higher methylation in certain gene upstream regions compared to 'Hayward'

  • Similar epigenetic variations might affect P5CS genes

• Potential regulatory mechanisms:

  • Promoter methylation affecting transcription factor binding

  • Histone modifications influencing chromatin accessibility

  • Small RNA-directed regulation of P5CS expression

  • Stress-induced epigenetic changes affecting P5CS regulation

• Research approaches:

  • Bisulfite sequencing to profile DNA methylation across P5CS loci

  • ChIP-seq for histone modification analysis at P5CS regulatory regions

  • RNA-seq to identify potential regulatory non-coding RNAs

  • Correlation of epigenetic marks with P5CS expression and proline levels

Understanding epigenetic regulation could reveal how different cultivars have adapted to environmental conditions and provide targets for epigenetic manipulation to enhance desired traits .