Recombinant Populus trichocarpa Methylthioribose-1-phosphate isomerase (POPTRDRAFT_832064)

What is the biochemical function of Methylthioribose-1-phosphate isomerase in Populus trichocarpa?

Methylthioribose-1-phosphate isomerase (encoded by POPTRDRAFT_832064 in Populus trichocarpa) catalyzes the conversion of methylthioribose-1-phosphate (MTR-1-P) to methylthioribulose-1-phosphate (MTRu-1-P) in the methionine salvage pathway. This isomerization reaction represents a critical step in the recycling of methionine, which serves as a precursor for S-adenosylmethionine (SAM) - a universal methyl donor in transmethylation reactions and a precursor for ethylene, polyamine, and phytosiderophore biosynthesis .

To study this function experimentally, researchers commonly employ recombinant protein expression systems, enzyme activity assays measuring the conversion rate of MTR-1-P to MTRu-1-P, and gene knockout/knockdown approaches to observe phenotypic effects. Isotope labeling with 35S-methionine can also be used to track methionine recycling flux through this pathway.

How is POPTRDRAFT_832064 structurally characterized and how does this compare to homologs in other species?

The POPTRDRAFT_832064 gene encodes a 375 amino acid protein that belongs to the eIF-2B alpha/beta/delta subunits family, specifically the MtnA subfamily . Structural characterization typically involves:

• Protein crystallization and X-ray diffraction analysis

• Homology modeling based on related structures

• Circular dichroism spectroscopy to determine secondary structure content

• Size-exclusion chromatography to determine quaternary structure

A comparative analysis approach involves aligning the Populus trichocarpa sequence with homologs from other plant species, bacteria, and fungi using tools like CLUSTALW followed by phylogenetic analysis. This can reveal conserved catalytic residues and structural features. For methylthioribose-1-phosphate isomerases, the core structure typically contains a triose-phosphate isomerase (TIM) barrel fold, which is characteristic of many isomerases .

What expression systems are most effective for producing recombinant POPTRDRAFT_832064?

When expressing POPTRDRAFT_832064, researchers should consider incorporating a histidine tag or other fusion partners to facilitate purification. For optimal expression in E. coli, codon optimization may be necessary due to differences in codon usage between Populus trichocarpa and E. coli. Expression at lower temperatures (16-25°C) often improves solubility of plant enzymes in bacterial systems.

How does methylthioribose-1-phosphate isomerase activity relate to abiotic stress response in Populus trichocarpa?

The methionine recycling pathway, in which methylthioribose-1-phosphate isomerase plays a key role, is intimately connected to ethylene biosynthesis and polyamine production - both crucial for plant stress responses . To investigate this relationship:

• Perform quantitative RT-PCR analysis of POPTRDRAFT_832064 expression under various stress conditions (drought, salinity, temperature extremes)

• Create transgenic poplar lines with altered POPTRDRAFT_832064 expression levels

• Compare metabolite profiles (focusing on methionine, SAM, and polyamines) between wild-type and transgenic plants under stress

• Measure ethylene production under stress conditions, as the methionine recycling pathway affects ethylene synthesis capacity

Research has shown that in plants like rice, the upregulation of methionine recycling genes (including S-adenosylmethionine synthetase, methylthioribose kinase, and acireductone dioxygenase 1) occurs during bacterial colonization, suggesting a role in plant-microbe interactions . Similar mechanisms may exist in Populus trichocarpa, making this enzyme relevant to both biotic and abiotic stress responses.

What are the kinetic parameters of recombinant POPTRDRAFT_832064 and how do they compare to orthologs?

Kinetic characterization of recombinant POPTRDRAFT_832064 requires:

• Expression and purification of the enzyme to >95% homogeneity

• Development of a reliable assay for measuring isomerase activity

• Determination of Km, Vmax, kcat, and kcat/Km values under varying conditions:

For comparative analysis with orthologs, identical experimental conditions must be maintained across all enzyme preparations. Differences in kinetic parameters may reflect evolutionary adaptations to specific environmental niches or metabolic demands within different plant species. Additionally, investigate the effects of potential inhibitors, activators, and the metal ion dependence of the enzyme activity.

How does site-directed mutagenesis of conserved residues affect POPTRDRAFT_832064 activity and substrate specificity?

Based on structural alignment with characterized methylthioribose-1-phosphate isomerases and the typical TIM barrel fold structure , several conserved residues likely play critical roles in substrate binding and catalysis. To investigate:

• Identify conserved residues through multiple sequence alignment of methylthioribose-1-phosphate isomerases across diverse species

• Perform site-directed mutagenesis on:

  • Catalytic residues (typically acidic or basic amino acids)

  • Substrate binding pocket residues

  • Structural residues maintaining the TIM barrel fold

• Express and purify mutant proteins

• Evaluate enzyme kinetics and compare with wild-type enzyme

• Test substrate specificity by assaying activity with structural analogs of MTR-1-P

Expected impacts of mutations include:

• Changes in Km (reflecting altered substrate binding affinity)

• Reduced kcat (indicating compromised catalytic efficiency)

• Altered pH optimum (suggesting changed protonation states of catalytic residues)

• Modified substrate specificity (potentially creating novel biocatalytic applications)

What are the optimal conditions for assaying Methylthioribose-1-phosphate isomerase activity?

Designing a robust assay for methylthioribose-1-phosphate isomerase requires careful optimization of several parameters:

The activity can be monitored through:

• Direct measurement of MTRu-1-P formation using HPLC

• Coupled enzymatic assays that link product formation to NAD(P)H oxidation/reduction

• NMR spectroscopy to observe substrate-to-product conversion in real-time

• Isothermal titration calorimetry to measure reaction thermodynamics

The assay should include appropriate controls:

• Heat-inactivated enzyme

• Reaction without substrate

• Reaction without cofactors

• Standard curves for substrate and product quantification

How can CRISPR-Cas9 genome editing be applied to study POPTRDRAFT_832064 function in Populus trichocarpa?

CRISPR-Cas9 genome editing offers powerful approaches to study gene function in plants, though applying it to woody species like Populus trichocarpa presents unique challenges:

• sgRNA design:

  • Target specific exons of POPTRDRAFT_832064

  • Consider using multiple sgRNAs to increase editing efficiency

  • Verify specificity using genome databases to avoid off-target effects

• Delivery methods:

  • Agrobacterium-mediated transformation of leaf discs or stem segments

  • Particle bombardment of embryogenic callus

  • Protoplast transformation followed by regeneration

• Mutation strategies:

  • Gene knockout through frameshift mutations

  • Domain-specific modifications

  • Promoter editing to alter expression levels

  • Precise base editing for specific amino acid substitutions

• Phenotypic analysis of edited lines:

  • Growth characteristics and morphology

  • Methionine content and recycling efficiency

  • Metabolomic profiling focusing on SAM-derived metabolites

  • Stress response testing (particularly ethylene-mediated responses)

A complementation approach, where the wild-type gene is reintroduced into knockout lines, should be employed to confirm phenotype-genotype correlations and rule out off-target effects.

What approaches can be used to study the spatiotemporal expression patterns of POPTRDRAFT_832064 in Populus trichocarpa?

Understanding where and when POPTRDRAFT_832064 is expressed provides crucial insights into its physiological role in Populus trichocarpa:

• Transcriptional analysis:

  • Quantitative RT-PCR of different tissues and developmental stages

  • RNA-Seq of tissue-specific transcriptomes

  • Single-cell RNA-Seq for cellular resolution

• Protein localization:

  • Generation of antibodies against recombinant POPTRDRAFT_832064

  • Immunohistochemistry of tissue sections

  • Creation of GFP fusion proteins for in vivo localization

  • Subcellular fractionation followed by western blotting

• Promoter analysis:

  • Cloning the native promoter region upstream of reporter genes (GUS, GFP)

  • Transgenic expression in Populus to visualize activity patterns

  • Identification of regulatory elements through deletion analysis

  • ChIP-Seq to identify transcription factors binding to the promoter

• Temporal dynamics:

  • Analysis across developmental stages from seedling to mature tree

  • Response to diurnal cycles

  • Seasonal variations, particularly in relation to dormancy

  • Stress-induced expression changes

This multi-faceted approach enables correlation of expression patterns with physiological processes and environmental responses, providing context for the enzyme's role in plant metabolism.

How should contradictory results between in vitro enzyme activity and in vivo phenotypes be reconciled?

Discrepancies between in vitro biochemical data and in vivo observations are common in enzyme research and require systematic investigation:

• Potential sources of contradiction:

  • Post-translational modifications present in vivo but absent in recombinant protein

  • Metabolic channeling effects not replicated in purified enzyme systems

  • Regulatory interactions with other proteins or metabolites

  • Compartmentalization effects in cellular environments

  • Differences in substrate availability or concentrations

• Resolution approaches:

  • Perform enzyme assays with native protein extracted from Populus tissues

  • Recreate cellular conditions more accurately (pH, ion concentrations, macromolecular crowding)

  • Use isotope labeling to track metabolic flux through the pathway in vivo

  • Identify and characterize protein interaction partners

  • Create targeted point mutations that affect activity in vitro and test in transgenic plants

• Integrative analysis:

  • Systems biology modeling of the methionine recycling pathway

  • Multi-omics approaches combining transcriptomics, proteomics, and metabolomics

  • Comparative studies across multiple plant species with varying MTRI activities

When interpreting contradictory results, consider that in vitro conditions represent an isolated system optimized for measuring specific parameters, while in vivo environments involve complex regulatory networks and competing reactions.

What computational approaches can predict substrate binding and catalytic mechanisms of POPTRDRAFT_832064?

Computational methods provide valuable insights into enzyme mechanism and can guide experimental design:

• Homology modeling and structure prediction:

  • Generate 3D models based on crystal structures of related isomerases

  • Refine models using molecular dynamics simulations

  • Validate structural predictions through experimental approaches (CD spectroscopy, limited proteolysis)

• Molecular docking studies:

  • Dock MTR-1-P and analogs into the active site

  • Identify key residues involved in substrate recognition

  • Calculate binding energies and compare with experimental Km values

• Quantum mechanics/molecular mechanics (QM/MM) simulations:

  • Model the reaction coordinate from substrate to product

  • Calculate energy barriers for catalytic steps

  • Predict effects of mutations on transition state stabilization

• Molecular dynamics simulations:

  • Analyze protein flexibility and conformational changes

  • Investigate water networks in the active site

  • Study allosteric effects and protein-protein interactions

These computational predictions should be validated experimentally through site-directed mutagenesis, enzyme kinetics, and structural studies. The predictions can also guide the design of inhibitors or substrate analogs for further mechanistic studies.

How can understanding POPTRDRAFT_832064 contribute to improving bioenergy production in Populus species?

Methylthioribose-1-phosphate isomerase's role in methionine recycling connects it to several pathways relevant to bioenergy applications:

Understanding this enzyme's role in tree metabolism could lead to poplar varieties with improved growth characteristics, modified lignin content for easier processing, and enhanced tolerance to environmental stresses - all critical factors for sustainable bioenergy production.

What role might POPTRDRAFT_832064 play in plant-microbe interactions in the Populus rhizosphere?

The methionine recycling pathway has been implicated in plant-microbe interactions, particularly in nitrogen-fixing symbioses and plant responses to bacterial colonization:

• Evidence from other systems:

  • Studies in rice have shown that methionine recycling genes (including S-adenosylmethionine synthetase, methylthioribose kinase, and acireductone dioxygenase 1) are upregulated during bacterial colonization

  • Ethylene production, influenced by methionine recycling, modulates plant-microbe interactions

  • Phytosiderophore synthesis, dependent on the methionine cycle, affects rhizosphere microbial communities

• Research approaches:

  • Transcriptomic analysis of POPTRDRAFT_832064 expression during colonization by beneficial microbes

  • Metabolomic analysis of rhizosphere exudates from wild-type and transgenic Populus

  • Co-culture experiments with varying POPTRDRAFT_832064 expression levels

  • Field studies examining rhizosphere microbial communities in relationship to enzyme activity

• Potential mechanisms:

  • Altered root exudate composition affecting microbial recruitment

  • Modified ethylene signaling changing plant immune responses

  • Improved nutrient acquisition through enhanced methionine recycling supporting symbionts

This research direction connects plant biochemistry with ecosystem-level interactions, potentially revealing how fundamental metabolic processes influence complex biological communities in the rhizosphere.