GDA Human
Description
Molecular Structure and Production
GDA Human is produced as a recombinant protein in Escherichia coli systems. Key structural features include:
The enzyme belongs to the ATZ/TRZ family of metallo-dependent hydrolases and contains 454–477 amino acids depending on the isoform. Its active site facilitates the hydrolytic deamination of guanine to xanthine and ammonia, a reaction critical for purine catabolism .
Enzymatic Activity and Substrate Specificity
GDA Human catalyzes guanine deamination with a reaction half-life (t<sub>1/2</sub>) of 27–31 seconds under optimal conditions . Recent studies using emissive guanine surrogates revealed:
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Isothiazolo guanine (tzG<sup>N</sup>): Fully deaminated by GDA with kinetics comparable to native guanine (t<sub>1/2</sub> = 29 s).
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Thieno guanine (thG<sup>N</sup>): Resistant to enzymatic conversion, highlighting the importance of N7 and O6 positions for substrate recognition .
Inhibition assays identified AICA (IC<sub>50</sub> = 100 μM) and ATCA (IC<sub>50</sub> = 80 μM) as competitive inhibitors, providing tools for modulating GDA activity in disease models .
Neuronal Development
GDA promotes microtubule assembly in neuronal dendrites by interacting with PSD-95, a postsynaptic scaffolding protein. Dysregulation is linked to:
Skin Pigmentation
In epidermal keratinocytes, GDA upregulates stem cell factor (SCF) and endothelin-1 (ET-1), enhancing melanogenesis. Elevated GDA levels correlate with hyperpigmented lesions in conditions like Riehl’s melanosis .
Metabolic Regulation
By converting guanine to xanthine, GDA prevents hyperuricemia and gout. Its activity reduces guanine nucleotide pools, indirectly lowering uric acid production .
Diagnostic Tools
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Real-time activity assays: Emissive substrates like tzG<sup>N</sup> enable fluorescence-based monitoring of GDA activity .
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High-throughput screening: Used to identify inhibitors for metabolic or neurological disorders .
Therapeutic Targets
| Condition | Mechanism | Reference |
|---|---|---|
| Gout/Hyperuricemia | Reduces guanine-derived uric acid | |
| Melanoma/Skin Disorders | Modulates SCF/ET-1 signaling | |
| Neurodegeneration | Regulates dendritic microtubules |
Key Research Findings
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Structural Insights: The N-terminal His-tag in ENZ-982 does not impair enzymatic activity, enabling affinity purification without functional loss .
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Inhibitor Development: ATCA, a novel isothiazole inhibitor, shows comparable efficacy to AICA, expanding the chemical toolbox for GDA modulation .
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Clinical Correlations: Serum ET-1 and SCF levels are significantly elevated in patients with GDA-associated hyperpigmentation (p < 0.05) .
Product Specs
Q&A
What is the primary enzymatic function of GDA in human cellular physiology?
GDA catalyzes the irreversible deamination of guanine to xanthine, a critical step in purine catabolism. Methodological verification requires:
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Spectrophotometric assays: Monitor absorbance at 249 nm (xanthine formation) using 50 mM Tris-HCl buffer (pH 8.0) at 37°C .
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HPLC validation: Separate reaction products via C18 column (mobile phase: 20 mM KH2PO4, pH 3.5) .
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Ammonia detection: Couple with glutamate dehydrogenase reaction system .
Table 1: Recombinant GDA catalytic parameters
| Parameter | Value (Source) |
|---|---|
| Specific activity | >2,000 pmol/min/μg |
| Km (guanine) | 18.7 μM (calculated from ) |
| Optimal pH | 8.0–8.5 |
| Thermal stability | 4°C stable ≥4 weeks |
What structural features enable recombinant human GDA production in E. coli?
The 53 kDa protein (477 amino acids, UniProt Q9HCP7) contains:
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N-terminal His-tag: 23 residues for nickel-affinity purification
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Thermostabilizing elements: 10% glycerol in storage buffer preserves activity
Experimental validation protocol:
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SDS-PAGE: Confirm >90% purity via 12% gel (Coomassie staining)
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Circular dichroism: Verify secondary structure integrity (α-helix content ≥40%)
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Dynamic light scattering: Ensure monodisperse preparation (PDI <0.2)
How to optimize GDA activity assays for tissue homogenates?
Address interference using:
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Precipitation steps: 10% TCA treatment removes contaminating proteins
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Inhibitor cocktails: 1 mM EDTA chelates endogenous metal ions
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Blank correction: Subtract background xanthine oxidase activity
How to design CRISPR-KO studies investigating GDA’s role in nucleotide imbalance disorders?
Multiparametric approach:
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sgRNA design: Target exon 3 (5'-GCACCGAGTCGGTGCTCAAC-3')
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Phenotypic screening:
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Rescue experiments: Transfect catalytically active mutant (D153A)
Table 2: Genomic integration strategies from GDA tool
| Data Type | Analytical Method | Output Metrics |
|---|---|---|
| Drug response | Spearman correlation (ρ) | IC50 vs. GDA expression |
| Mutation | Fisher’s exact test | Mutation frequency in responders |
| Expression | WGCNA network analysis | Co-expressed gene modules |
What computational models reconcile contradictory substrate specificity reports?
Address discrepancies via:
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Molecular dynamics simulations:
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100 ns trajectories in CHARMM36m force field
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Compare guanine vs. 8-oxoguanine binding affinities
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Free energy perturbation: Calculate ΔΔG using AMBER20
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Experimental validation: Synthesize GDA-K294R mutant to test predicted contact residues
How to implement single-cell GDA activity profiling in tumor biopsies?
Multi-omics pipeline:
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Microfluidic separation: 10 μm channels capture individual cells
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Kinetic readout: Fluorogenic probe 5-nitro-6-aminouracil (λex/em=380/450 nm)
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Correlative analysis:
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scRNA-seq for pathway activation signatures
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MALDI-TOF MS for metabolite zonation
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Technical considerations:
What cryo-EM advancements enable high-resolution GDA oligomerization studies?
Recent 1.8 Å structures reveal:
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Sample preparation:
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3 mM CHAPSO in vitrification buffer
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30 s blot time at 100% humidity
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Validation workflow:
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SEC-MALS: Confirm 106 kDa dimeric state
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Hydrogen-deuterium exchange: Map dynamic regions
How to standardize GDA biomarker validation across multi-center cohorts?
Consensus guidelines:
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Pre-analytical variables:
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Plasma collection: Streck Cell-Free DNA BCT tubes
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Processing delay: <2 hr at 4°C
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Assay harmonization:
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Reference material: NIST SRM 1953
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QC criteria: ≤15% CV across replicates
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Product Science Overview
GDA is a cytoplasmic enzyme found in various tissues, including the brain, liver, and kidneys. The enzyme’s primary function is to convert guanine into xanthine, which is then further metabolized into uric acid by xanthine oxidase . This process is vital for maintaining the balance of purine nucleotides within the cell and preventing the accumulation of toxic guanine levels.
The human GDA enzyme has a molecular mass of approximately 51 kDa and is typically found as a dimer . The enzyme’s activity is optimal at a pH of around 7.0, and it shares a conserved sequence motif with other aminohydrolases and amidohydrolases .
Recombinant human GDA is produced by cloning the GDA gene into an expression vector, such as the pMAL vector, and expressing the protein in a suitable host system like Escherichia coli . The recombinant protein is then purified using affinity chromatography techniques, ensuring high purity and activity.
The recombinant GDA enzyme retains its ability to catalyze the conversion of guanine to xanthine with a Michaelis constant (Km) of approximately 9.5 µM . This value is consistent with the Km values reported for other mammalian guanine deaminases, which range from 4.2 to 15.3 µM .
GDA activity is essential for the proper functioning of the purine metabolism pathway. In cases of xanthine dehydrogenase deficiency, the conversion of guanine to xanthine becomes even more critical, as it helps maintain the balance of purine nucleotides . Additionally, GDA has been implicated in various physiological processes, including neuronal development and function .
Deficiencies or abnormalities in GDA activity can lead to the accumulation of guanine and its derivatives, resulting in various pathological conditions. For example, lead-induced inactivation of GDA activity has been associated with the deposition of guanine crystals in the epiphyseal plate of the femoral head . Moreover, a deficiency in brain GDA activity has been reported in a full-term infant who presented with acute anoxia and died from respiratory distress .
