Recombinant Trichoplax adhaerens Probable methylthioribulose-1-phosphate dehydratase (TRIADDRAFT_64275)
Description
Enzymatic Role and Pathway Context
Methylthioribulose-1-phosphate dehydratase (MtnB) catalyzes the dehydration of 5-methylthioribulose-1-phosphate (MTRu-1-P) to 2,3-diketo-5-methylthiopentyl-1-phosphate (DK-MTP-1-P) in the methionine salvage pathway . This pathway recycles 5'-methylthioadenosine (MTA), a byproduct of polyamine synthesis, back into methionine, critical for cellular proliferation and redox balance . In Trichoplax adhaerens, TRIADDRAFT_64275 is annotated as a putative MtnB, suggesting a conserved metabolic role .
Table 1: Comparative Features of MtnB Homologs
Functional Implications in Trichoplax adhaerens
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Methionine Salvage: Likely essential for recycling MTA in this basal metazoan, given its minimalistic genome and reliance on conserved metabolic pathways .
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Post-Translational Modifications: Proteomic studies of T. adhaerens reveal high tyrosine phosphorylation activity, hinting at regulatory mechanisms that may influence MtnB function .
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Evolutionary Conservation: TRIADDRAFT_64275 shares sequence homology with human APIP/MtnB (e.g., zinc-binding residues), suggesting ancient origin of this enzymatic function .
Research Gaps and Future Directions
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Enzymatic Assays: Kinetic parameters (, ) for TRIADDRAFT_64275 remain uncharacterized .
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Structural Studies: X-ray crystallography or cryo-EM is needed to resolve its 3D architecture.
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Biological Context: Functional links to apoptosis or inflammation, as observed in human APIP/MtnB , are unexplored in T. adhaerens.
References to Key Studies
Product Specs
Target Background
KEGG: tad:TRIADDRAFT_64275
STRING: 10228.TriadP64275
Q&A
What is TRIADDRAFT_64275 and why is it significant for research?
TRIADDRAFT_64275 refers to the gene encoding a probable methylthioribulose-1-phosphate dehydratase in Trichoplax adhaerens, one of the evolutionarily earliest diverging and simplest living animals. This enzyme is predicted to participate in the methionine salvage pathway, similar to characterized methylthioribulose-1-phosphate dehydratases in other organisms. Its significance lies in understanding primitive metabolic pathways in one of the earliest metazoans.
T. adhaerens belongs to the phylum Placozoa, inhabits subtropical and tropical waters, and consists of only six cell types. As a model organism, it offers unique insights into early animal evolution due to its simple morphology and phylogenetic position dating back to the Precambrian era . Studies on T. adhaerens proteins reveal adaptations to environments with low oxygen levels (approximately 8 mg O₂/L compared to air's 210 mg O₂/L) and limited nutrient availability . These environmental factors likely shaped distinctive enzymatic properties in T. adhaerens metabolism.
What is the predicted function of methylthioribulose-1-phosphate dehydratase in metabolic pathways?
Based on characterized orthologs, methylthioribulose-1-phosphate dehydratase likely catalyzes the conversion of 5-methylthioribulose-1-phosphate (MTRu-1-P) to 2,3-diketo-5-methylthiopentyl-1-phosphate (DK-MTP-1-P) in the methionine salvage pathway . This pathway recycles the sulfur-containing portion of methionine, an essential amino acid, maintaining cellular methionine homeostasis.
In Bacillus subtilis, where this enzyme has been thoroughly characterized, methylthioribulose-1-phosphate dehydratase functions as a tetrameric protein with a molecular mass of approximately 90 kDa . The enzyme demonstrates specific kinetic properties with a Km of 8.9 μM and Vmax of 42.7 μmol min⁻¹ mg protein⁻¹ at 25°C, optimal activity at pH 7.5-8.5, and temperature optimum at 40°C . The activation energy for the reaction is 63.5 kJ mol⁻¹ . Notably, the reaction product DK-MTP-1-P is unstable, decomposing with a rate constant of 0.048 s⁻¹ to compounds not utilized by downstream enzymes . In T. adhaerens, the enzyme likely performs a similar function but may exhibit different properties adapted to marine environments.
How does T. adhaerens serve as a model organism for studying primitive enzymatic functions?
T. adhaerens represents an excellent model for studying primitive enzymatic functions due to several key characteristics. It possesses a relatively small genome containing fundamental metabolic pathway components, including various enzymes essential for basic cellular processes . Proteomic studies have demonstrated that T. adhaerens expresses numerous metabolic enzymes at quantifiable levels, enabling functional investigations .
The organism's living conditions—marine environments with low oxygen content and limited nutrient availability—provide context for understanding enzymatic adaptations in early metazoans. For instance, T. adhaerens experiences low cobalamin availability (approximately 2 pM in seawater) and reduced oxygen levels compared to terrestrial environments . These conditions have likely driven selective pressures for enzymatic features optimized for such environments.
Comparative analyses between T. adhaerens enzymes and homologs from more complex organisms offer insights into evolutionary refinement of enzymatic functions. For example, the T. adhaerens cobalamin processing enzyme (TaCblC) exhibits distinct functional characteristics compared to human orthologs, including differences in electron transfer coupling and substrate processing rates, reflecting adaptations to its unique cellular environment .
What expression systems are optimal for recombinant production of TRIADDRAFT_64275?
Several expression systems could be employed for recombinant production of TRIADDRAFT_64275, each with specific advantages and limitations:
The choice of system should consider the protein's characteristics. For initial characterization, E. coli would typically be the starting point, as successfully implemented for other T. adhaerens proteins like TaCblC . Various fusion tags can be employed to enhance solubility and facilitate purification, including His, FLAG, MBP, GST, trxA, Nus, or GFP tags, with positioning at either the N- or C-terminus .
For optimal expression, codon optimization aligned with the chosen expression system is recommended, particularly given the evolutionary distance between T. adhaerens and common expression hosts . A pilot expression study comparing different systems, conditions, and fusion tags would be advisable to determine the optimal approach.
What purification strategies are most effective for recombinant TRIADDRAFT_64275?
An effective purification strategy for recombinant TRIADDRAFT_64275 would typically involve multiple chromatographic steps, potentially including:
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Affinity chromatography: If expressed with a fusion tag (His, MBP, GST), corresponding affinity resins provide efficient initial capture . For His-tagged proteins, immobilized metal affinity chromatography (IMAC) using Ni-NTA resin is commonly employed.
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Ion exchange chromatography: Based on the predicted isoelectric point (pI) of the protein, cation or anion exchange can be used for further purification.
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Size exclusion chromatography: Particularly important if the native enzyme forms multimeric structures (likely tetrameric based on the B. subtilis ortholog) .
The following table outlines potential purification conditions:
| Purification Step | Buffer Considerations | Parameter Optimization |
|---|---|---|
| Cell lysis | pH 7.5-8.5, 20-50 mM buffer, 100-300 mM NaCl, protease inhibitors | Mechanical vs. chemical lysis methods |
| Affinity chromatography | Imidazole gradient for His-tag (20-300 mM) | Binding, washing, and elution conditions |
| Ion exchange | pH based on protein pI, 20-500 mM salt gradient | pH and conductivity optimization |
| Size exclusion | PBS or similar, flow rate optimization | Molecular weight determination |
| Tag removal | Specific protease (TEV, thrombin, etc.) | Cleavage conditions, re-purification |
Quality control testing should include SDS-PAGE (>90% purity), Western blot (identity confirmation), and activity assays (functional verification) . Additional protein reprocessing steps might include renaturation (if expressed as inclusion bodies), endotoxin removal, filtration sterilization, and lyophilization .
How might the structure of T. adhaerens methylthioribulose-1-phosphate dehydratase compare to characterized orthologs?
While specific structural information for TRIADDRAFT_64275 is not directly available, comparative analysis with characterized methylthioribulose-1-phosphate dehydratases provides insights into potential structural features:
| Structural Feature | Potential Adaptation in T. adhaerens | Functional Implication |
|---|---|---|
| Oligomeric assembly | Potentially tetrameric with modified interfaces | Stability in marine conditions |
| Active site architecture | Conserved catalytic residues with modified substrate binding pocket | Substrate specificity/affinity |
| Surface properties | Altered surface charge distribution | Adaptation to ionic strength of marine environment |
| Flexible regions | Potentially more flexible at lower temperatures | Activity at lower environmental temperatures |
| Stability elements | Structural adaptations for oxygen-limited environments | Altered redox sensitivity |
Drawing parallels from studies on TaCblC, which showed structural adaptations compared to human orthologs (e.g., Tyr170 in TaCblC vs. Ser146 in human CblC), TRIADDRAFT_64275 may similarly possess unique residues that contribute to its specialized function in T. adhaerens . These adaptations might reflect environmental pressures of the marine habitat, including temperature, oxygen availability, and nutrient limitations.
What kinetic parameters would be expected for recombinant TRIADDRAFT_64275?
Based on the characterized B. subtilis ortholog and considering T. adhaerens' environmental conditions, the following kinetic parameters might be anticipated for TRIADDRAFT_64275:
| Kinetic Parameter | B. subtilis Enzyme Value | Predicted Range for T. adhaerens Enzyme | Rationale for Prediction |
|---|---|---|---|
| Km | 8.9 μM | 5-15 μM | Similar substrate recognition with potential adaptations |
| Vmax | 42.7 μmol min⁻¹ mg⁻¹ | 15-45 μmol min⁻¹ mg⁻¹ | Potentially lower due to adaptation to cooler environments |
| Optimal pH | 7.5-8.5 | 7.0-8.0 | Adaptation to slightly different intracellular pH in marine environment |
| Optimal temperature | 40°C | 20-30°C | Likely optimized for marine temperatures |
| Activation energy | 63.5 kJ mol⁻¹ | 50-70 kJ mol⁻¹ | Core chemistry likely similar |
| Product stability | DK-MTP-1-P decays at 0.048 s⁻¹ | Potentially different decay rate | May have evolved mechanisms to stabilize the product |
The enzyme's behavior under varying oxygen conditions would be particularly interesting to investigate, given T. adhaerens' adaptation to lower oxygen environments compared to terrestrial organisms . The stability and activity profiles might reflect adaptations to these conditions, potentially showing different electron transfer coupling efficiency and redox sensitivity compared to orthologs from aerobic organisms.
How does environmental oxygen level affect enzyme function in T. adhaerens proteins?
T. adhaerens inhabits marine environments with significantly lower oxygen levels than terrestrial habitats (approximately 8 mg O₂/L compared to air's 210 mg O₂/L) . This environmental constraint likely shaped the evolutionary adaptations of T. adhaerens enzymes, including potential adaptations in TRIADDRAFT_64275.
Studies on the cobalamin processing enzyme of T. adhaerens (TaCblC) provide insights into how proteins from this organism may be adapted to lower oxygen environments. TaCblC exhibits distinct functional characteristics compared to human orthologs:
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Enhanced reaction rates: TaCblC dealkylates methylcobalamin approximately 2-times faster than human CblC .
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Different product stabilization: TaCblC stabilizes cob(II)alamin under aerobic conditions, unlike human CblC .
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Altered electron transfer coupling: TaCblC shows higher unproductive electron transfer with increased glutathione oxidation .
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Structural adaptations: Specific residues (e.g., Tyr170 in TaCblC vs. Ser146 in human CblC) create a more hydrophobic environment near reactive centers, potentially impeding water binding and influencing redox chemistry .
These observations suggest that TRIADDRAFT_64275 might similarly exhibit distinct functional properties reflecting adaptation to lower oxygen environments. This could include altered redox sensitivity, different coupling efficiency in electron transfer reactions, and structural features that function optimally under the specific oxygen tensions experienced by T. adhaerens.
What are the optimal assay conditions for measuring TRIADDRAFT_64275 activity?
Based on characterized methylthioribulose-1-phosphate dehydratases and T. adhaerens' environmental conditions, the following assay parameters would be recommended:
Activity measurement methods could include:
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Direct assay: Monitoring substrate depletion or product formation by HPLC or LC-MS.
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Coupled assay: Linking the reaction to a detectable change (e.g., spectrophotometric).
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End-point analysis: Quenching reactions at various time points for product quantification.
Given the reported instability of DK-MTP-1-P (decomposition rate constant of 0.048 s⁻¹ in the B. subtilis system) , rapid analysis methods or stabilization strategies might be necessary. Time-course experiments under various oxygen concentrations would be particularly valuable, given the potential oxygen sensitivity of T. adhaerens enzymes .
How can site-directed mutagenesis inform structure-function relationships in TRIADDRAFT_64275?
Site-directed mutagenesis represents a powerful approach to investigate structure-function relationships in TRIADDRAFT_64275. A systematic mutagenesis strategy would target:
| Residue Category | Mutation Strategy | Expected Outcome | Analysis Methods |
|---|---|---|---|
| Catalytic residues | Alanine substitution | Reduced/abolished activity | Activity assays, kinetic analysis |
| Substrate binding | Conservative substitutions | Altered Km, substrate specificity | Substrate binding studies, kinetic analysis |
| Oligomerization interface | Interface-disrupting mutations | Altered quaternary structure | Size exclusion chromatography, native PAGE |
| Redox-sensitive residues | Cysteine/methionine substitutions | Changed oxygen sensitivity | Activity under varying O₂ conditions |
| Unique T. adhaerens residues | Substitution with residues from terrestrial orthologs | Modified environmental adaptation | Comparative activity under different conditions |
The mutagenesis approach should be informed by:
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Multiple sequence alignment of methylthioribulose-1-phosphate dehydratases from diverse organisms to identify conserved catalytic residues and lineage-specific substitutions.
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Homology modeling based on available crystal structures of related enzymes to predict the three-dimensional context of target residues.
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Comparative analysis with characterized mutations in related enzymes, such as the findings for TaCblC where specific residues (e.g., Tyr170) were identified as potentially important for unique functional properties .
A particularly informative approach would be to create chimeric proteins, swapping domains between the T. adhaerens enzyme and terrestrial orthologs to identify regions responsible for environmental adaptations. This could reveal how evolutionary pressures shaped enzymatic function in this primitive metazoan.
What approaches can resolve contradictory activity data for TRIADDRAFT_64275?
When confronted with contradictory activity data for TRIADDRAFT_64275, systematic troubleshooting approaches can help identify sources of variability:
| Source of Contradiction | Investigation Approach | Resolution Strategy |
|---|---|---|
| Protein preparation variability | Circular dichroism spectroscopy, size exclusion chromatography | Standardize expression and purification protocols |
| Substrate quality/stability | HPLC/MS analysis of substrate | Implement rigorous substrate quality control |
| Oxygen sensitivity | Activity assays under defined O₂ concentrations | Standardize oxygen conditions, potentially using anaerobic techniques |
| Metal ion dependency | Activity with/without EDTA, metal reconstitution series | Define metal requirements and standardize metal content |
| Redox state fluctuations | Controlled redox potential, varying reducing agents | Establish optimal redox conditions |
| Buffer components effects | Systematic buffer component screening | Identify optimal buffer composition |
| Product detection issues | Multiple detection methods, kinetic analysis | Validate assay methodology |
As observed with TaCblC, proteins from T. adhaerens may exhibit unexpected properties related to oxygen sensitivity and electron transfer coupling . For TRIADDRAFT_64275, activity patterns might similarly vary depending on experimental conditions, particularly oxygen levels. Careful control and documentation of all experimental variables would be essential.
A comprehensive experimental design would include:
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Replicate measurements under identical conditions to establish reproducibility
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Systematic variation of individual parameters to identify critical factors
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Multiple detection methods to verify results through orthogonal approaches
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Detailed documentation of all experimental conditions and protein preparation methods
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Comparative analysis with orthologs from different organisms under identical conditions
Such systematic approaches would help resolve contradictions and establish reliable protocols for consistent characterization of TRIADDRAFT_64275 activity.
How should researchers address protein instability issues with recombinant TRIADDRAFT_64275?
Potential instability of recombinant TRIADDRAFT_64275 can be addressed using several strategies:
| Stability Issue | Solution Approach | Implementation Strategy | Evaluation Method |
|---|---|---|---|
| Low expression yield | Codon optimization | Gene synthesis with host-optimized codons | Quantitative protein yield comparison |
| Inclusion body formation | Fusion partners | MBP, GST, or SUMO tag incorporation | Soluble fraction analysis by SDS-PAGE |
| Expression temperature | Reduce to 15-18°C during induction | Soluble vs. insoluble protein ratio | |
| Solubilizing additives | Screen osmolytes, detergents, arginine | Activity retention after purification | |
| Proteolytic degradation | Protease inhibitors | Comprehensive inhibitor cocktail | SDS-PAGE time course stability |
| Construct optimization | Remove flexible regions via truncation | Limited proteolysis mapping | |
| Oxidative damage | Anaerobic handling | Oxygen-free buffers and handling | Activity retention over time |
| Reducing agents | DTT, β-ME, TCEP at optimal concentrations | Thiol status monitoring | |
| Thermal instability | Buffer optimization | Screen stabilizing additives/conditions | Differential scanning fluorimetry |
| Storage conditions | Test cryoprotectants, lyophilization | Activity retention after storage |
T. adhaerens proteins may show adaptation to cooler marine environments, potentially resulting in thermal instability when expressed recombinantly. The TaCblC protein demonstrated reduced thermal stability compared to human orthologs , suggesting TRIADDRAFT_64275 might exhibit similar characteristics.
A systematic approach to stability optimization would include:
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Initial stability profiling using differential scanning fluorimetry to identify destabilizing conditions
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Thermal shift assays with various buffer components to identify stabilizing additives
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Storage stability tests under different conditions (temperature, additives, concentration)
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Activity assays before and after stress conditions to assess functional stability
For long-term storage, lyophilization in the presence of appropriate stabilizers might be considered, although this approach requires validation to ensure activity retention after reconstitution .
What challenges arise when comparing TRIADDRAFT_64275 with orthologs from evolutionarily distant organisms?
Comparative analysis of TRIADDRAFT_64275 with orthologs from diverse organisms presents several challenges that require careful experimental approaches:
The T. adhaerens enzyme likely exhibits adaptations to specific environmental conditions including lower oxygen levels, lower temperatures, and marine ionic composition . These adaptations may manifest as altered kinetic parameters, substrate specificity, or stability profiles compared to orthologs from terrestrial organisms.
A robust comparative approach would include:
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Phylogenetic analysis to establish evolutionary relationships among orthologs
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Parallel expression and purification of multiple orthologs using identical methods
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Activity characterization under standardized conditions as well as organism-specific optimal conditions
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Structural analysis through comparative modeling and, ideally, experimental structure determination
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Identification of lineage-specific substitutions that correlate with functional differences
As demonstrated in the comparative analysis of TaCblC with human orthologs, such approaches can reveal significant insights into the evolution of enzymatic function and environmental adaptation .
