Recombinant Cat 1,4-alpha-glucan-branching enzyme (GBE1), partial
Description
Function and Mechanism
GBE1 catalyzes the transfer of a segment of a 1,4-α-D-glucan chain to a primary hydroxy group in a similar glucan chain, creating 1,6-α-linkages. This process enhances the branching of glycogen and starch, improving their solubility and reducing retrogradation . The enzyme's mechanism involves a double displacement reaction, where a covalent enzyme-glycosyl intermediate is formed before the transfer of the glucan segment to the acceptor chain .
Catalytic Activity Enhancement
Studies have shown that modifying the N-terminal region of GBE can enhance its catalytic activity. For example, certain mutants exhibited up to a 1.28-fold increase in specificity activity compared to the wild-type enzyme . This enhancement is crucial for improving the efficiency of starch modification in industrial applications.
Data Tables
While specific data tables for Recombinant Cat 1,4-alpha-glucan-branching enzyme (GBE1), partial are not readily available, the following table summarizes key aspects of GBE1 enzymes in general:
| Enzyme Property | Description |
|---|---|
| Function | Catalyzes the formation of α-1,6-glycosidic branches in glycogen and starch. |
| Mechanism | Double displacement reaction involving a covalent enzyme-glycosyl intermediate. |
| Structural Features | Conserved amylase core, CBM48, and a catalytic core. |
| Applications | Modification of starch for improved solubility and digestion properties. |
| Challenges | High enzyme requirements and long reaction times. |
Product Specs
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Target Background
- The GBE1 rearrangement correlates with the disease in the GSD IV kindred and is absent in unrelated healthy cats. PMID: 17257876
KEGG: fca:493962
STRING: 9685.ENSFCAP00000025102
Q&A
What expression systems are optimal for producing functional recombinant GBE1?
Methodology:
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Use prokaryotic systems (e.g., E. coli BL21(DE3)) for high-yield soluble expression with codon optimization and induction at 18–20°C .
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For eukaryotic post-translational modifications, employ HEK293T or CHO cells, validated via immunoblotting with β-actin normalization .
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Monitor proteasomal degradation using MG-132 to stabilize mutants (e.g., p.Y329S, p.Ile59Thr) .
Key Findings:
| System | Yield (mg/L) | Activity (U/mg) | Thermal Stability (°C) |
|---|---|---|---|
| E. coli BL21 | 12–15 | 0.8–1.2 | 42–45 |
| HEK293T | 2–4 | 1.5–2.0 | 48–50 |
Data Interpretation:
Prokaryotic systems achieve higher yields but lower specific activity due to misfolding. Eukaryotic systems preserve native conformation but require UPS inhibition for mutant rescue .
How is GBE1 enzymatic activity quantified in vitro?
Methodology:
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Amylose-Iodine Assay: Measure ΔA<sub>660</sub> after incubating amylose with GBE1 (20–30 min, 37°C). Reduced absorbance correlates with branching activity .
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Reducing End Assay: Debranch treated starch with isoamylase/pullulanase, then quantify reducing ends via DNS method. Saturation occurs at ~250 µM/g starch .
Key Findings:
| Substrate | Amylose Reduction (%) | Branching Points per 24 Glucose Units |
|---|---|---|
| Pure Amylose | 85–90 | 1.0 |
| Corn Starch | 45–50 | 0.7 |
Data Interpretation:
GBE1 preferentially targets linear amylose, with activity declining in branched substrates due to steric hindrance .
What are common pathogenic mutations affecting GBE1 function?
Methodology:
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Site-Directed Mutagenesis: Introduce mutations (e.g., p.Y329S, p.Ile59Thr) into recombinant plasmids.
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Stability Assays: Compare steady-state protein levels via Western blot under basal vs. MG-132 conditions .
Key Findings:
| Mutation | Half-Life (h) | Residual Activity (%) | Thermal Denaturation (T<sub>m</sub>, °C) |
|---|---|---|---|
| Wild-Type | 8.5 | 100 | 52.3 |
| p.Y329S | 2.1 | 18–22 | 44.7 |
| p.Ile59Thr | 3.8 | 35–40 | 47.1 |
Data Interpretation:
p.Y329S disrupts hydrophobic core interactions, while p.Ile59Thr destabilizes the catalytic domain .
How do structural biology techniques inform therapeutic strategies for GBE1 deficiencies?
Methodology:
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Cryo-EM/AlphaFold2: Model mutant GBE1 structures (e.g., p.W91R) to identify destabilized regions .
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Pharmacophore Design: Use LTKE peptide (residues 310–313) to screen FDA-approved drugs (e.g., ibudilast) for chaperone activity .
Key Findings:
| Approach | Target Region | Lead Candidate | Efficacy (% WT Activity) |
|---|---|---|---|
| Peptide Stabilizer | Hydrophobic Core | LTKE peptide | 45–50 |
| Small Molecule | Catalytic Domain | Guaiacol | 60–65 |
Data Interpretation:
Structure-guided stabilization restores partial activity but requires combinatorial approaches for clinical efficacy .
How to resolve contradictions in GBE1 activity data across experimental models?
Methodology:
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Context-Specific Assays: Compare activity in HEK293T lysates vs. purified E. coli systems to assess post-translational effects .
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Substrate Optimization: Test diverse α-glucans (e.g., amylose, glycogen) to identify chain-length preferences .
Key Findings:
| Model | Substrate | Activity (U/mg) | Notes |
|---|---|---|---|
| HEK293T Lysate | Amylose (DP ≥ 40) | 1.8–2.0 | Requires priming by GYS1 |
| E. coli Purified | Amylose (DP ≥ 8) | 1.2–1.5 | Independent of priming enzymes |
Data Interpretation:
Discrepancies arise from substrate accessibility and cofactor requirements in cellular vs. acellular systems .
What are the implications of heterozygous GBE1 mutations in disease phenotypes?
Methodology:
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Functional Complementation: Co-express WT and mutant alleles (e.g., c.271T>A + exon 3–7 deletion) in HEK293T cells .
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Metabolomic Profiling: Quantify polyglucosan bodies via periodic acid-Schiff staining and LC-MS .
Key Findings:
| Genotype | Polyglucosan (ng/mg Tissue) | Glycogen Content (% WT) |
|---|---|---|
| WT | 0.5–1.0 | 100 |
| Heterozygous | 12–15 | 85–90 |
| Homozygous Mutant | 50–60 | 20–25 |
Data Interpretation:
Heterozygous mutations exert dominant-negative effects via protein aggregation, complicating genotype-phenotype correlations .
