Recombinant Vigna aconitifolia Delta-1-pyrroline-5-carboxylate synthase, partial

What is the molecular structure and organization of Vigna aconitifolia P5CS?

Vigna aconitifolia P5CS is a bifunctional enzyme with a native molecular mass of 450 kDa composed of six identical subunits. The enzyme exhibits two distinct catalytic activities: gamma-glutamyl kinase (γ-GK) and glutamic acid-5-semialdehyde (GSA) dehydrogenase. These activities catalyze the first two steps in proline biosynthesis, converting glutamate to GSA, which spontaneously cyclizes to form Delta-1-pyrroline-5-carboxylate (P5C) . Structurally, the enzyme contains domains with significant homology to bacterial enzymes, with conserved regions for substrate binding and catalysis . The bifunctional nature of plant P5CS differs from bacteria, where these activities are typically encoded by separate genes .

What are the kinetic parameters of the wild-type Vigna aconitifolia P5CS?

The wild-type Vigna aconitifolia P5CS demonstrates the following kinetic parameters:

• Km for glutamate: 3.6 mM

• Km for ATP: 2.7 mM

• 50% inhibition of γ-GK activity occurs at approximately 5 mM proline

• The GSA dehydrogenase activity is not sensitive to proline inhibition

These parameters indicate moderate affinity for its substrates and significant sensitivity to feedback inhibition by proline, particularly affecting the γ-GK activity while the GSA dehydrogenase activity remains unaffected by proline levels .

How does P5CS contribute to plant stress response mechanisms?

P5CS plays a central role in plant stress response by catalyzing the rate-limiting step in proline biosynthesis. Under osmotic stress conditions such as drought or high salinity, plants upregulate P5CS expression, leading to increased proline accumulation . Northern blot analysis reveals that P5CS transcript levels in Vigna aconitifolia roots are enhanced when plants are treated with 200 mM NaCl . Proline serves as an osmolyte, helping maintain cellular water potential, and also functions as a protective agent for macromolecules during stress conditions . The regulation of P5CS and proline dehydrogenase during stress and recovery periods controls proline levels in response to environmental conditions .

What expression systems are most effective for recombinant Vigna aconitifolia P5CS production?

Escherichia coli has proven to be an effective expression system for recombinant Vigna aconitifolia P5CS. The P5CS cDNA can be successfully expressed in E. coli, as demonstrated in multiple studies . For expression, the cDNA can be cloned into appropriate expression vectors (such as pET-based vectors) and transformed into E. coli strains. Complementation assays with E. coli proB, proA, and proBA mutants (proline auxotrophs) can be used to verify functional expression . Expression can be optimized by adjusting induction conditions (IPTG concentration, temperature, and duration) to maximize protein yield while maintaining enzymatic activity.

What purification strategy yields the highest purity and activity for recombinant P5CS?

A multi-step purification approach is recommended for obtaining homogeneous P5CS with high activity:

• Ammonium sulfate fractionation: This initial step helps concentrate the protein and remove some contaminants

• Ion-exchange chromatography: Cation exchange chromatography, such as carboxymethyl (CM) cellulose, can be effective for P5CS purification

• Gel filtration: Analytical gel filtration chromatography on columns such as Sephadex G-200 can be used for final purification and determination of native molecular weight

This strategy has been shown to yield purified enzyme with preserved bifunctional activity. The purification process should be monitored using SDS-PAGE and enzyme activity assays to ensure retention of both γ-GK and GSA dehydrogenase activities .

How can the enzymatic activities of purified P5CS be accurately measured?

The bifunctional activity of P5CS can be measured using distinct assays for each catalytic function:

For γ-GK activity:

• Hydroxamate assay: Measures the formation of γ-glutamyl hydroxamate

• [14C]glutamate assay: Tracks the phosphorylation of radiolabeled glutamate

• Inhibition studies: γ-GK activity can be measured as a function of proline concentration to assess feedback inhibition

For GSA dehydrogenase activity:

• NADPH oxidation: Monitored spectrophotometrically at 340 nm

• P5C formation: Can be detected using specific colorimetric assays

Enzymatic activity should be expressed as specific activity (units per mg protein) and compared with published values for quality control .

How is P5CS activity regulated in plants at the molecular level?

P5CS activity in plants is regulated through multiple mechanisms:

• Feedback inhibition: The γ-GK activity of P5CS is competitively inhibited by proline, the end product of the pathway. This inhibition occurs with 50% inhibition at approximately 5 mM proline for the wild-type enzyme .

• Transcriptional regulation: P5CS gene expression is enhanced under osmotic stress conditions. Northern blot analysis shows increased transcript levels in Vigna roots under salt stress .

• Protein inhibitor: A protein inhibitor of P5CS has been observed in plant cells, suggesting an additional layer of regulation .

• Reciprocal regulation with proline degradation: A coordinated increase in P5CS and decrease in proline dehydrogenase during stress conditions, with the reverse occurring during recovery, helps maintain appropriate proline levels .

What methods are available to study the feedback inhibition of P5CS by proline?

Several methodological approaches can be used to study the feedback inhibition of P5CS by proline:

• Enzymatic assays with varying proline concentrations: γ-GK activity can be measured as a function of proline concentration (ranging from 0.01 to 100 mM) to determine inhibition kinetics and calculate IC50 values .

• Site-directed mutagenesis: Specific amino acid residues implicated in proline binding can be mutated to analyze their role in feedback inhibition .

• Comparative studies: The behavior of wild-type P5CS can be compared with mutant variants (such as P5CSF129A) to evaluate differences in feedback sensitivity .

• Structural analysis: Molecular modeling based on structural data can help identify the proline-binding site and understand the mechanism of inhibition .

These approaches have revealed that the γ-GK activity is specifically inhibited by proline, while the GSA dehydrogenase activity remains unaffected .

What is the significance of the F129A mutation in modifying feedback inhibition?

The F129A mutation (substitution of phenylalanine at position 129 with alanine) in Vigna aconitifolia P5CS significantly reduces the enzyme's sensitivity to proline feedback inhibition. Key findings include:

• The 50% inhibition values for γ-GK activity increase dramatically from 5 mM proline in the wild-type to 960 mM proline in the mutant P5CSF129A .

• Transgenic plants expressing P5CSF129A accumulate approximately 2-fold more proline than plants expressing wild-type P5CS under both normal and stressed conditions .

• The mutation specifically affects feedback inhibition without altering other enzymatic properties .

This mutation demonstrates that targeted modifications can effectively reduce allosteric regulation, providing a valuable tool for enhancing proline biosynthesis and potentially improving stress tolerance in plants .

How can recombinant P5CS variants be used to study proline metabolism in plants?

Recombinant P5CS variants offer powerful tools for studying proline metabolism:

What experimental approaches can determine if P5CS maintains feedback regulation under stress conditions?

Determining whether P5CS maintains feedback regulation under stress conditions requires several experimental approaches:

• Transgenic comparison studies: Plants expressing wild-type P5CS can be compared with those expressing feedback-insensitive P5CSF129A under both normal and stressed conditions. If P5CS retains feedback regulation during stress, plants with the mutant enzyme should accumulate significantly more proline than those with wild-type P5CS during stress .

• In vitro enzyme assays: Protein extracts from stressed and non-stressed plants can be analyzed for P5CS activity and sensitivity to proline inhibition .

• Proline accumulation kinetics: Monitoring the rate and extent of proline accumulation in response to stress in plants with different P5CS variants can provide insights into regulatory mechanisms .

Results from these approaches have demonstrated that feedback regulation of P5CS is not completely eliminated under stress conditions, as transgenic plants expressing P5CSF129A accumulate approximately 2-fold more proline than those expressing wild-type P5CS even under stress .

How can recombinant P5CS be utilized in metabolic engineering for enhanced stress tolerance?

Recombinant P5CS can be strategically employed in metabolic engineering to enhance plant stress tolerance:

• Expression of feedback-insensitive variants: Introducing P5CSF129A into plants can increase proline accumulation and potentially enhance tolerance to osmotic stress .

• Tissue-specific or stress-inducible expression: Using appropriate promoters to drive P5CS expression in specific tissues or under stress conditions can optimize proline accumulation while minimizing potential negative effects on growth and development .

• Combination with other stress tolerance genes: P5CS modifications can be combined with other stress tolerance mechanisms for synergistic effects .

• Cross-species application: The conserved nature of P5CS across plant species suggests that strategies developed using Vigna aconitifolia P5CS could be applied to other agriculturally important crops .

These approaches leverage the understanding gained from basic research on P5CS structure and function to develop practical applications for improving crop resilience to environmental stresses .

What spectroscopic methods are most suitable for structural characterization of recombinant P5CS?

For structural characterization of recombinant P5CS, several spectroscopic methods are particularly suitable:

• Circular Dichroism (CD) spectroscopy: Useful for assessing secondary structure content and conformational changes upon substrate binding or in response to mutations.

• Fluorescence spectroscopy: Can be employed to study protein folding, tertiary structure, and binding interactions with substrates or inhibitors by monitoring intrinsic tryptophan fluorescence or using fluorescent probes.

• FTIR spectroscopy: Provides information about secondary structure elements and can detect subtle changes in protein conformation.

• NMR spectroscopy: For detailed structural analysis of smaller domains or fragments of P5CS.

• X-ray crystallography: The gold standard for high-resolution structural determination, though challenging for large, multidomain proteins like P5CS .

These techniques can provide complementary information about structural features that influence enzyme activity, substrate binding, and allosteric regulation .

How can mass spectrometry be applied to study post-translational modifications of P5CS?

Mass spectrometry offers powerful approaches for studying post-translational modifications (PTMs) of P5CS:

• Bottom-up proteomics: Enzymatic digestion of purified P5CS followed by LC-MS/MS analysis can identify PTMs such as phosphorylation, acetylation, or oxidation that may regulate enzyme activity.

• Top-down proteomics: Analysis of intact P5CS can reveal the combinatorial patterns of multiple PTMs and their stoichiometry.

• Targeted MS approaches: Multiple reaction monitoring (MRM) or parallel reaction monitoring (PRM) can quantify specific PTMs across different conditions or treatments.

• Cross-linking MS: Can identify interaction sites between domains or with regulatory proteins that may influence enzyme function.

These approaches can reveal how PTMs might change in response to stress conditions and potentially regulate P5CS activity or stability, providing insights beyond the primary sequence information .

What computational methods can predict the impact of mutations on P5CS structure and function?

Several computational methods can be employed to predict how mutations might affect P5CS structure and function:

• Homology modeling: Building structural models based on related proteins with known structures, particularly useful since complete P5CS structures are not yet available .

• Molecular dynamics simulations: Simulating the dynamic behavior of wild-type and mutant P5CS to understand how mutations affect protein flexibility, stability, and domain interactions.

• Molecular docking: Predicting how mutations might alter substrate binding, catalysis, or allosteric regulation by proline.

• Sequence conservation analysis: Identifying evolutionarily conserved residues that may be critical for function.

• Energy calculations: Assessing how mutations might affect protein stability or the energetics of substrate binding.

These computational approaches can guide experimental design by identifying promising mutation targets for enhancing enzyme properties or understanding naturally occurring variants .

How does Vigna aconitifolia P5CS compare with P5CS enzymes from other plant species?

Comparing Vigna aconitifolia P5CS with enzymes from other plant species reveals significant conservation along with some species-specific differences:

• Structural conservation: The bifunctional nature of P5CS, combining γ-GK and GSA dehydrogenase activities, appears to be conserved across plant species including Arabidopsis . This is distinct from bacterial systems where these activities are encoded by separate genes .

• Sequence homology: Alignment of P5CS sequences from different species reveals high conservation, particularly in catalytic and regulatory domains .

• Regulatory mechanisms: The feedback inhibition by proline is a conserved feature, though the sensitivity to inhibition may vary between species .

• Isoforms: Some plant species possess multiple P5CS isoforms with different expression patterns or regulatory properties, while others may have a single isoform .

This evolutionary conservation underscores the fundamental importance of P5CS in plant metabolism and stress adaptation .

What insights can be gained from comparing plant P5CS with bacterial gamma-glutamyl kinase and glutamic-5-semialdehyde dehydrogenase?

Comparative analysis of plant P5CS with bacterial counterparts provides valuable insights:

• Evolutionary fusion: Plant P5CS represents a fusion of two bacterial enzymes (ProB and ProA) into a single bifunctional enzyme, suggesting evolutionary pressure for coordinated regulation of these sequential reactions .

• Regulatory conservation: The aspartate residue implicated in proline feedback inhibition in bacterial γ-GK is conserved in plant P5CS, indicating conservation of regulatory mechanisms across diverse organisms .

• Structural adaptations: Despite functional similarities, plant P5CS has unique structural features that may reflect adaptations to plant-specific metabolic needs and regulatory networks .

• Oligomeric organization: The hexameric structure of Vigna P5CS (450 kDa with six identical subunits) differs from bacterial γ-GK, suggesting potential differences in allosteric regulation mechanisms .

These comparisons illuminate evolutionary relationships and can guide efforts to engineer P5CS with desired properties based on knowledge from bacterial systems .

How has P5CS evolved to function in different environmental conditions across plant species?

The evolution of P5CS across plant species reflects adaptations to diverse environmental challenges:

• Stress sensitivity: Plants from different ecological niches show variations in P5CS expression patterns and sensitivity to environmental triggers, reflecting adaptation to their typical stress exposures .

• Regulatory mechanisms: Fine-tuning of feedback inhibition and transcriptional regulation may vary across species, potentially correlating with their natural habitat conditions and stress tolerance strategies .

• Tissue-specific expression: The pattern of P5CS expression across different tissues may vary between species, suggesting specialized roles in development or stress response .

• Isoform diversification: Some species have evolved multiple P5CS isoforms with specialized functions or expression patterns, allowing for more nuanced responses to environmental challenges .

Understanding these evolutionary adaptations provides insights into natural strategies for stress tolerance that can inform biotechnological approaches to crop improvement .