Recombinant Danio rerio Methylthioribose-1-phosphate isomerase (mri1)

What is Methylthioribose-1-phosphate isomerase (mri1) and its functional role in zebrafish metabolism?

Methylthioribose-1-phosphate isomerase (mri1) is a critical enzyme in the methionine salvage pathway (MSP) that catalyzes the conversion of methylthioribose-1-phosphate (MTR-1-P) to methylthioribulose-1-phosphate (MTRu-1-P). In zebrafish, as in other vertebrates, this enzyme plays an essential role in recycling sulfur from methylthioadenosine, a byproduct of polyamine synthesis, back to methionine. The zebrafish genome contains highly conserved orthologous genes with humans (~70% orthology), making it an excellent model system for studying this metabolic pathway . The conserved nature of this enzyme across species suggests its fundamental importance in cellular metabolism.

How does zebrafish mri1 compare structurally and functionally to human MRI1?

The zebrafish mri1 protein shares significant structural and functional homology with its human counterpart. Based on comparative analyses of related methylthioribose-1-phosphate isomerases, we can infer that:

Structural conservation between zebrafish and human MRI1 makes zebrafish an appropriate model for studying the functional impacts of human MRI1 mutations.

How is mri1 integrated into the methionine salvage pathway in zebrafish?

In the zebrafish methionine salvage pathway, mri1 functions as an intermediary enzyme that follows 5'-methylthioadenosine phosphorylase (MTAP) activity. The pathway operates in the following sequence:

• 5'-methylthioadenosine (MTA) is converted to methylthioribose-1-phosphate (MTR-1-P) by MTAP

• MTR-1-P is isomerized to methylthioribulose-1-phosphate (MTRu-1-P) by mri1

• MTRu-1-P undergoes dehydration by MtnB enzymes to form diketomethylthiopentene-1-phosphate (DK-MTP-1-P)

• Further reactions ultimately lead to the regeneration of methionine

In some organisms, fusion proteins like MtnBD, containing both dehydratase and dioxygenase functions, have been identified in this pathway . Such fusion proteins can catalyze multiple sequential reactions of the pathway, though their presence in zebrafish has not been explicitly documented in the provided search results.

What expression systems are most effective for producing recombinant Danio rerio mri1?

Based on similar recombinant protein expression protocols for zebrafish enzymes and methylthioribose-1-phosphate isomerases from other species, the following expression systems have proven effective:

Optimal expression typically involves:

• Cloning the full-length zebrafish mri1 cDNA into a vector with an appropriate fusion tag (His6, GST, or MBP)

• Transformation into the chosen expression strain

• Induction with IPTG (typically 0.1-0.5 mM) at lower temperatures (16-25°C) for 16-20 hours to maximize soluble protein yield

• Incorporation of protease inhibitors during cell lysis to prevent degradation

What purification strategies yield the highest activity for recombinant zebrafish mri1?

A multi-step purification approach is recommended to obtain high-purity, active recombinant mri1:

• Initial capture: Affinity chromatography using Ni2+-NTA for His-tagged proteins or glutathione-agarose for GST-tagged proteins

• Intermediate purification: Ion exchange chromatography (typically Q-Sepharose) to remove contaminating proteins

• Polishing step: Size exclusion chromatography to obtain homogeneous protein preparation and to determine the oligomeric state of the enzyme

Buffer optimization is crucial for maintaining enzyme activity. A typical buffer system would include:

• 50 mM Tris-HCl or HEPES, pH 7.5-8.0

• 100-150 mM NaCl

• 1-5 mM DTT or 2-mercaptoethanol

• 10% glycerol for storage stability

• Optional: 0.1-1 mM divalent metal ions (Mg2+ or Mn2+) if required for activity

How can researchers address protein solubility challenges when expressing recombinant Danio rerio mri1?

Solubility issues are common when expressing recombinant proteins. For zebrafish mri1, the following strategies have proven effective for similar enzymes:

• Temperature optimization: Lowering induction temperature to 16-25°C significantly improves solubility for many fish proteins

• Fusion tags: Solubility-enhancing tags such as MBP, SUMO, or Thioredoxin can dramatically improve soluble expression

• Co-expression with chaperones: GroEL/GroES chaperone co-expression systems can help with protein folding

• Buffer optimization: Addition of mild solubilizing agents during lysis:

  • 5-10% glycerol

  • 0.1% Triton X-100

  • 50-100 mM L-arginine

  • 5-10 mM β-mercaptoethanol to maintain reduced cysteine residues

• Codon optimization: Adjusting codons for optimal expression in the host organism, particularly for rare codons in zebrafish genes expressed in E. coli

What enzymatic assays are most reliable for measuring mri1 activity in vitro?

Several complementary assays can be used to assess recombinant zebrafish mri1 activity:

• Spectrophotometric coupled enzyme assay:

  • Principles: The isomerization reaction is coupled to subsequent enzymes in the pathway, with the consumption of NAD(P)H monitored at 340 nm

  • Advantages: Real-time monitoring, quantitative kinetic parameters

  • Limitations: Requires additional purified enzymes, potential for interference

• 1H-NMR spectroscopy:

  • Principles: Direct observation of substrate conversion based on chemical shift changes

  • Advantages: Direct visualization of reaction intermediates without coupling enzymes

  • Applications: Especially useful for confirming the formation of methylthioribulose-1-phosphate from methylthioribose-1-phosphate

• HPLC/LC-MS analysis:

  • Principles: Separation and quantification of substrate and product

  • Advantages: Highly sensitive, can detect multiple reaction intermediates

  • Recommended setup: Reverse-phase HPLC with UV detection or MS detection for enhanced sensitivity

For optimal results, reaction conditions typically include:

• 50 mM Tris-HCl or HEPES buffer, pH 7.5-8.0

• 5 mM MgCl2

• 1-2 mM DTT

• 0.1-1 mM methylthioribose-1-phosphate substrate

• Temperature: 25-30°C (or 37°C for comparative studies with mammalian enzymes)

How can isotope labeling be implemented to track mri1 activity in the methionine salvage pathway?

Isotope labeling provides powerful insights into the reaction mechanism and metabolic flux through the methionine salvage pathway:

• Deuterium incorporation studies:

  • Using D2O as the reaction solvent allows tracking of proton exchange during the reaction

  • 1H-NMR analysis can reveal deuterium incorporation at specific carbon positions

  • This approach has been successfully used to elucidate the reaction mechanism of related isomerases, showing deuterium incorporation at the C4 position

• 13C-labeled substrates:

  • Synthesizing methylthioribose-1-phosphate with 13C at specific positions

  • Following the fate of labeled carbons through the pathway using 13C-NMR

  • Particularly useful for determining reaction stereochemistry and identifying transient intermediates

• 35S-labeled methionine:

  • For in vivo or cell-based systems, tracking the recycling of sulfur through the pathway

  • Can be combined with autoradiography or scintillation counting for quantification

What spectroscopic methods can reveal the catalytic mechanism of zebrafish mri1?

Multiple spectroscopic approaches provide complementary insights into mri1 catalytic mechanisms:

• X-ray crystallography:

  • Determining the three-dimensional structure of zebrafish mri1 alone and in complex with substrate/product analogs

  • Identifying key active site residues through structural comparison with homologous enzymes

  • Crystallization typically requires protein at >95% purity and 5-10 mg/ml concentration

• Circular dichroism (CD) spectroscopy:

  • Monitoring secondary structure changes upon substrate binding

  • Temperature-dependent CD to assess thermal stability and the effect of ligands

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Probing protein dynamics and conformational changes during catalysis

  • Identifying regions with differential solvent accessibility upon substrate binding

• Site-directed mutagenesis coupled with activity assays:

  • Targeted mutation of predicted catalytic residues based on homology with related isomerases

  • Key residues likely include conserved histidines that may be essential for the isomerization reaction

  • Kinetic analysis of mutants to determine the role of specific residues in catalysis

How can morpholino knockdown be optimized to study mri1 function in zebrafish embryos?

Morpholino (MO) antisense oligonucleotides are effective tools for transient gene knockdown in zebrafish embryos. For mri1 studies:

• Morpholino design:

  • Translation-blocking MOs: Designed to target the region spanning the start codon

  • Splice-blocking MOs: Target exon-intron boundaries to disrupt normal splicing

  • Recommended to design 2-3 different MOs to control for off-target effects

• Optimal delivery protocol:

  • Microinjection into 1-4 cell stage embryos is the standard delivery method

  • Typical concentration range: 2-8 ng per embryo

  • Include phenol red (0.05%) for visualization during injection

  • Use fine-tipped glass needles (1-2 μm diameter) and a micromanipulator

• Controls and validation:

  • Standard control MO with 5 mismatches

  • Rescue experiments with co-injection of mri1 mRNA to confirm specificity

  • RT-PCR to verify splice-blocking efficiency or western blotting to confirm protein knockdown

• Phenotypic analysis:

  • Monitor development through 7 days post-fertilization (dpf)

  • Document phenotypes using brightfield and fluorescent microscopy

  • Quantitative measurements of morphological features and developmental timing

What phenotypes might emerge from disruption of mri1 in zebrafish development?

Based on the role of mri1 in the methionine salvage pathway and studies of related metabolic enzymes in zebrafish, potential phenotypes could include:

Since methionine is crucial for protein synthesis and methylation reactions, disruption of its recycling pathway through mri1 knockdown might affect:

How can the zebrafish mri1 model be leveraged to understand human metabolic disorders?

Zebrafish provide an excellent model system for translational research on human metabolic disorders:

• Complementation studies:

  • Injecting human MRI1 mRNA (wild-type or mutant variants) into mri1-deficient zebrafish embryos

  • Assessing the ability of human MRI1 to rescue phenotypes in zebrafish

  • This approach enables functional evaluation of potentially pathogenic human variants

• Metabolic profiling:

  • Targeted metabolomics focusing on methionine, homocysteine, and related metabolites

  • Untargeted metabolomics to identify novel metabolic perturbations

  • Comparison with metabolic signatures from human patients with suspected methionine cycle disorders

• Integration with human genomic data:

  • Using zebrafish models to functionally validate variants of uncertain significance identified in human patients

  • Prioritizing candidate genes from human studies for detailed functional characterization in zebrafish

• Drug screening and therapeutic development:

  • Testing compounds that might bypass or compensate for defects in the methionine salvage pathway

  • Small-molecule screens using zebrafish embryos with disrupted mri1 function

What CRISPR-Cas9 strategies are most effective for generating stable mri1 mutant zebrafish lines?

CRISPR-Cas9 genome editing offers advantages over morpholino knockdown for creating stable genetic models:

• gRNA design considerations:

  • Target early exons to maximize disruption of protein function

  • Design 2-3 gRNAs targeting different regions of the gene

  • Verify low off-target potential using prediction algorithms

  • Optimal gRNA length: 20 nucleotides plus PAM sequence (NGG)

• Delivery methods:

  • Co-injection of Cas9 mRNA (150-300 pg) and gRNA (25-50 pg) into 1-cell stage embryos

  • Alternative: Injection of ribonucleoprotein complexes (pre-assembled Cas9 protein and gRNA)

  • Include a fluorescent tracer to confirm successful injection

• Mutation screening and line establishment:

  • T7 endonuclease assay or heteroduplex mobility assay for initial screening

  • Sanger sequencing to identify specific mutations

  • Out-crossing F0 mosaic fish to establish stable F1 lines

  • Genotyping protocols for routine identification of mutant carriers

• Characterization approaches:

  • Allele-specific PCR for genotyping

  • qRT-PCR and western blotting to confirm reduction/absence of mri1 expression

  • Comprehensive phenotypic analysis with particular attention to metabolic parameters

How can advanced structural biology techniques be applied to understand zebrafish mri1 function?

Integrating structural biology with functional studies provides deeper insights into mri1 catalytic mechanisms:

How can systems biology approaches integrate mri1 function into broader metabolic networks?

Understanding mri1 in the context of global cellular metabolism requires integrative approaches:

• Metabolic flux analysis:

  • Using isotope-labeled precursors to trace carbon flow through connected pathways

  • 13C-methionine pulse-chase experiments in zebrafish embryos or cell lines

  • Mathematical modeling to quantify flux distribution and control points

• Multi-omics integration:

  • Combining transcriptomics, proteomics, and metabolomics data from mri1-deficient models

  • Network analysis to identify compensatory pathways and regulatory responses

  • Correlation analysis between metabolite levels and phenotypic outcomes

• Perturbation studies:

  • Chemical inhibition of related pathways to identify synthetic interactions

  • Nutritional manipulation (methionine restriction or supplementation)

  • Stress conditions to reveal conditional dependencies on the methionine salvage pathway

• Comparative systems analysis across species:

  • Comparing metabolic network organization in zebrafish, humans, and other model organisms

  • Identifying conserved and divergent aspects of methionine metabolism regulation

  • Leveraging evolutionary conservation to predict functional relationships