Recombinant Lithobates catesbeiana Probable methylthioribulose-1-phosphate dehydratase (apip)

How does APIP relate to amphibian metamorphosis and thyroid hormone signaling?

Amphibian metamorphosis is primarily regulated by thyroid hormones (THs), which trigger extensive gene expression reprogramming across tissues . While the search results don't directly link APIP to TH signaling in amphibians, we can infer potential relationships based on available data.

During metamorphosis, Lithobates catesbeiana undergoes substantial tissue remodeling, including lung maturation in preparation for air breathing . This process involves significant changes in transcriptional regulation. Key transcriptional regulators including thyroid hormone receptors α and β, TH-induced bZip protein, and CCAAT/enhancer binding protein 1 show increased transcript abundance during postembryonic development .

The connection between methionine metabolism and thyroid hormone signaling may be important during this period of intense biosynthetic activity and tissue remodeling. As metamorphosis progresses, tadpoles also show elevated levels of antioxidant enzymes such as catalase and Cu/Zn superoxide dismutase, revealing a requirement for increased antioxidant capacity following metamorphosis .

What are the established methods for assaying methylthioribulose-1-phosphate dehydratase activity?

Researchers studying methylthioribulose-1-phosphate dehydratase activity typically employ spectrophotometric assays that measure the increase in absorbance at 280 nm, which corresponds to the formation of the reaction product. Based on the literature, the following methodology has been established:

Michaelis-Menten kinetics assay:

• Prepare the substrate methylthioribulose-1-phosphate (MTRu-1-P) in a reaction mixture containing:

  • 50 mM Tris·HCl (pH 7.5)

  • 1 mM MgCl₂

  • 28 μg of bacterial methylthioribose-1-phosphate isomerase (MtnA)

  • Various concentrations of methylthioribose-1-phosphate (MTR-1-P)

• Calculate the exact MTRu-1-P concentration using the equilibrium constant between MTRu-1-P and MTR-1-P ([MTRu-1-P]/[MTR-1-P] = 6.0)

• Initiate the reaction by adding a limiting amount (0.3 μg in 0.2 μL) of the purified APIP/MtnB protein

• Monitor the reaction at 280 nm using a UV-Visible Spectrophotometer

• Calculate the concentration of the product, 2,3-dihydroxy-5-(methylthio)pent-1-ene-1-phosphate (HK-MTPenyl-1-P), using its molecular extinction coefficient, 9.5 × 10³ M⁻¹ cm⁻¹ at 280 nm

For comparing the activity of mutant enzymes to wild-type, researchers typically use the same procedure but with a standardized amount of enzyme (0.72 μg) .

How can recombinant Lithobates catesbeiana methylthioribulose-1-phosphate dehydratase be expressed and purified?

While the search results don't provide specific protocols for L. catesbeiana APIP expression, a general methodology can be adapted from similar enzyme expression systems:

Expression and purification protocol:

• Gene cloning:

  • Amplify the APIP gene from Lithobates catesbeiana cDNA using PCR

  • Insert the amplified gene into a suitable expression vector (e.g., pET16b) for bacterial expression with an N-terminal His-tag

• Protein expression:

  • Transform the expression vector into a bacterial expression strain such as E. coli Rosetta2 (DE3)

  • Induce protein expression with IPTG under optimized conditions (temperature, duration, concentration)

• Protein purification:

  • Harvest cells and lyse using appropriate methods (sonication, French press, etc.)

  • Purify the His-tagged protein using immobilized metal affinity chromatography

  • Perform size exclusion chromatography to improve purity

  • Confirm purity by SDS-PAGE and protein identity by Western blotting or mass spectrometry

What is the catalytic mechanism of methylthioribulose-1-phosphate dehydratase?

Based on structural studies and enzyme assays, a three-step catalytic mechanism has been proposed for methylthioribulose-1-phosphate dehydratase:

• Proton abstraction and enol formation: The conserved glutamic acid residue (Glu139 in the studied structure) acts as a catalytic acid/base to abstract a proton from C3 of MTRu-1-P. Simultaneously, the hydroxide is removed from C4 to coordinate with the zinc ion in the active site, resulting in enol formation .

• Tautomerization: The reaction proceeds with a thermodynamic slide from the enol to keto form through tautomerization, resulting in the formation of the di-keto product, 2,3-diketo-5-methylthiopentyl-1-phosphate (DK-MTP-1-P) .

• Product release: The glutamic acid residue donates a proton to the hydroxide ion, and the product DK-MTP-1-P diffuses out of the active site. Water molecules then return to the active site, coordinating with the zinc ion together with three histidine residues to form an octahedral coordination geometry .

The importance of the glutamic acid residue in this mechanism is supported by mutagenesis studies showing that mutation of this residue almost completely abolishes enzyme activity .

How does the structure of methylthioribulose-1-phosphate dehydratase contribute to its function?

The enzyme contains several key structural features that enable its catalytic function:

• Active site zinc coordination: The active site contains a zinc ion that is coordinated by three histidine residues in a tetrahedral geometry. In the substrate-free state, three water molecules complete the coordination sphere .

• Substrate binding pocket: The active site is structured to accommodate the methylthioribulose-1-phosphate substrate, with specific residues positioned to interact with different parts of the substrate molecule .

• Catalytic residues: Key residues are positioned to participate in the catalytic mechanism:

  • Glutamic acid (Glu139) acts as a catalytic acid/base

  • Lysine (Lys142) and asparagine (Asn166) form hydrogen bonds with Glu139, positioning it correctly for catalysis

  • Cysteine (Cys97) forms a hydrogen bond with O4 of the substrate

• Conservation of key residues: The catalytic residues (Glu139, Lys142, and Asn166) are absolutely conserved from bacteria to eukaryotes, highlighting their critical role in the enzyme's function .

How can site-directed mutagenesis be applied to study the function of key residues in Lithobates catesbeiana methylthioribulose-1-phosphate dehydratase?

Site-directed mutagenesis is a powerful technique for investigating the functional importance of specific amino acid residues in enzymes. For studying L. catesbeiana methylthioribulose-1-phosphate dehydratase, researchers can employ the following approach:

Site-directed mutagenesis protocol:

• Target residue identification:

  • Identify conserved residues through sequence alignment with homologous enzymes

  • Focus on residues in the active site, particularly those involved in substrate binding and catalysis

  • Prioritize the conserved glutamic acid, lysine, and asparagine residues that have been implicated in the catalytic mechanism

• Mutagenesis strategy:

  • Design mutagenic primers to introduce specific mutations

  • Perform PCR-based site-directed mutagenesis on the APIP expression construct

  • Verify mutations by DNA sequencing

• Functional analysis:

  • Express and purify the mutant proteins using the same protocol as for the wild-type enzyme

  • Measure enzyme activity using the spectrophotometric assay described earlier

  • Calculate relative activity compared to the wild-type enzyme

• Structural analysis:

  • If possible, determine the crystal structure of selected mutants to understand structural changes

  • Alternatively, perform molecular modeling to predict structural effects of mutations

This approach has been successfully used to demonstrate the critical role of Glu139 in the catalytic mechanism of related methylthioribulose-1-phosphate dehydratases .

What is the relationship between methylthioribulose-1-phosphate dehydratase activity and oxidative stress during amphibian metamorphosis?

While the search results don't directly connect methylthioribulose-1-phosphate dehydratase to oxidative stress in amphibians, we can explore potential relationships based on available data:

During amphibian metamorphosis, there is a significant increase in oxidative metabolism as tissues undergo remodeling. Research on Lithobates catesbeiana has shown that transcripts for antioxidant enzymes like catalase and Cu/Zn superoxide dismutase show elevated levels at the end of metamorphosis, indicating a requirement for increased antioxidant capacity .

The methionine salvage pathway, in which methylthioribulose-1-phosphate dehydratase participates, may intersect with cellular responses to oxidative stress in several ways:

• Methionine synthesis: By recycling methionine, the pathway ensures an adequate supply of this amino acid for protein synthesis during tissue remodeling .

• Connection to polyamine metabolism: Since the methionine salvage pathway recycles MTA produced during polyamine synthesis, it may indirectly influence polyamine levels, which have been implicated in stress responses .

• Potential dual role: APIP has been identified as both a methylthioribulose-1-phosphate dehydratase and an inhibitor of cell death . This dual role might be particularly relevant during metamorphosis, where programmed cell death and tissue remodeling occur extensively.

Future research could explore how changes in oxidative stress during metamorphosis affect APIP expression and activity, and conversely, how modulation of APIP activity might influence cellular responses to oxidative stress.

How does Lithobates catesbeiana methylthioribulose-1-phosphate dehydratase compare to homologous enzymes in other species?

A comparative analysis of methylthioribulose-1-phosphate dehydratase across species reveals important evolutionary insights:

The methionine salvage pathway is found in organisms from bacteria to plants and animals, indicating its ancient evolutionary origin and fundamental importance in cellular metabolism . While specific comparative data for L. catesbeiana methylthioribulose-1-phosphate dehydratase is not provided in the search results, general patterns can be inferred.

Key features that are likely conserved across species include:

• Catalytic mechanism: The basic mechanism involving a glutamic acid residue as a catalytic acid/base is likely conserved from bacteria to vertebrates, including amphibians .

• Active site architecture: The zinc coordination by three histidine residues and the positioning of key catalytic residues (glutamic acid, lysine, asparagine) are features that are highly conserved across diverse organisms .

• Substrate specificity: The enzyme's specificity for 5-methylthioribulose-1-phosphate is expected to be maintained across species, reflecting the conservation of the methionine salvage pathway .

A phylogenetic analysis of methylthioribulose-1-phosphate dehydratase sequences from different species would provide further insights into the evolutionary relationships and potential functional adaptations in different taxonomic groups.

What are the experimental considerations for comparing thyroid hormone response elements in the promoter regions of APIP across amphibian species?

For researchers interested in understanding how thyroid hormone regulation of APIP might vary across amphibian species, the following experimental approach is recommended:

Comparative promoter analysis protocol:

• Sequence acquisition and alignment:

  • Obtain genomic sequences containing the APIP gene and its upstream regulatory regions from multiple amphibian species

  • Align these sequences to identify conserved regions that might contain regulatory elements

• Thyroid hormone response element (TRE) identification:

  • Search for consensus TRE sequences (typically consisting of AGGTCA half-sites arranged as direct repeats, inverted repeats, or palindromes)

  • Use bioinformatic tools specifically designed to identify nuclear receptor binding sites

• Functional validation:

  • Clone the promoter regions from different species into reporter constructs

  • Transfect these constructs into appropriate cell lines

  • Treat with thyroid hormones (T3/T4) and measure reporter gene expression

  • Compare the responsiveness of promoters from different species

• Chromatin immunoprecipitation (ChIP):

  • Perform ChIP assays using antibodies against thyroid hormone receptors

  • Compare receptor binding to the APIP promoter regions across species

  • Identify species-specific differences in receptor occupancy

This approach would provide insights into how thyroid hormone regulation of APIP has evolved across amphibian species and potentially identify adaptations related to different metamorphic strategies or environmental conditions.

What are promising strategies for identifying small molecule modulators of methylthioribulose-1-phosphate dehydratase activity?

For researchers interested in identifying modulators of methylthioribulose-1-phosphate dehydratase activity, several approaches can be considered:

High-throughput screening strategy:

• Assay adaptation:

  • Miniaturize the spectrophotometric assay described earlier for use in 96- or 384-well plate format

  • Optimize conditions for stability and reproducibility

  • Validate the assay using known inhibitors or activators if available

• Compound library selection:

  • Choose appropriate libraries based on research goals (natural products, FDA-approved drugs, diversity-oriented libraries)

  • Consider in silico screening to prioritize compounds for testing

• Primary screening:

  • Test compounds at a single concentration

  • Identify hits showing significant inhibition or activation

  • Include appropriate controls to identify false positives

• Secondary screening:

  • Confirm hits in dose-response experiments

  • Determine IC50 or EC50 values

  • Assess selectivity against related enzymes

• Mechanistic characterization:

  • Determine mechanism of inhibition (competitive, non-competitive, uncompetitive)

  • Use site-directed mutagenesis to identify binding sites

  • Perform structural studies (crystallography, molecular docking) to understand binding modes

This approach would identify compounds that could serve as research tools for studying the methionine salvage pathway or potentially lead to the development of modulators with therapeutic applications.

How might CRISPR-Cas9 genome editing be used to investigate the role of methylthioribulose-1-phosphate dehydratase in amphibian development?

CRISPR-Cas9 genome editing offers powerful tools for investigating gene function in developmental biology. For studying methylthioribulose-1-phosphate dehydratase in amphibian development, researchers could employ the following strategy:

CRISPR-Cas9 experimental design:

• Target design:

  • Design guide RNAs targeting the APIP gene in Lithobates catesbeiana or other experimentally tractable amphibian species

  • Include controls targeting non-essential genes

  • Consider creating specific mutations (e.g., in the catalytic glutamic acid residue) rather than complete gene knockout

• Delivery method:

  • Microinject Cas9 protein and guide RNAs into fertilized eggs

  • Alternatively, electroporate Cas9-guide RNA ribonucleoprotein complexes into embryos

• Genotyping:

  • Develop PCR-based genotyping assays to identify successful editing events

  • Sequence targeted regions to confirm the nature of the induced mutations

• Phenotypic analysis:

  • Examine effects on embryonic development and metamorphosis

  • Monitor thyroid hormone responsiveness

  • Assess methionine metabolism using tracer studies

  • Measure polyamine levels and oxidative stress markers

• Rescue experiments:

  • Attempt to rescue any observed phenotypes by providing exogenous methionine or related metabolites

  • Introduce wild-type or mutant versions of the APIP gene to assess functional complementation

This approach would provide direct evidence for the role of methylthioribulose-1-phosphate dehydratase in amphibian development and potentially reveal new functions or regulatory mechanisms not apparent from biochemical studies alone.