GDA Human, His

What is human Guanine Deaminase and what is its primary function?

Human Guanine Deaminase (GDA) is an enzyme responsible for the hydrolytic deamination of guanine, converting it to xanthine through the removal of an amino group. This conversion represents a critical step in purine metabolism. The gene is located at chromosome 9q21.13, and multiple transcript variants encoding different isoforms have been identified .

Beyond its primary catalytic function, studies of the rat ortholog suggest GDA plays a role in microtubule assembly, indicating potential structural functions beyond metabolism . The enzyme is also known by several alternative names, including guanine aminase, guanine aminohydrolase, cytoplasmic PSD95 interactor, CYPIN, GUANASE, and NEDASIN .

What are the key structural characteristics of recombinant human GDA with histidine tag?

Recombinant human GDA with histidine tag typically has the following structural characteristics:

The recombinant protein contains the full human GDA sequence with an N-terminal histidine tag that facilitates purification using affinity chromatography. The amino acid sequence includes the MGSSHHHHHH SSGLVPRGSH MGS leader sequence containing the histidine tag followed by the human GDA sequence .

How can researchers verify the activity of purified recombinant human GDA?

Verifying the enzymatic activity of purified GDA is essential before proceeding with experiments. The specific activity of human GDA can be measured and should be >4,000 pmol/min/μg, defined as the amount of enzyme that converts guanine to xanthine per minute at pH 8.0 at 37°C .

Recently developed real-time monitoring methods utilize emissive heterocyclic cores as surrogate substrates. Unlike the thieno analog (thGN), the isothiazolo guanine surrogate (tzGN) undergoes effective enzymatic deamination by GDA and yields the spectroscopically distinct xanthine analog (tzX) . This spectral difference between substrate and product enables continuous monitoring of the reaction, providing a more robust assessment of enzyme activity than traditional endpoint assays.

What are the optimal storage conditions for maintaining GDA stability?

To maintain optimal stability and activity of recombinant human GDA with histidine tag, researchers should follow these storage guidelines:

• Short-term storage (1-2 weeks): Can be stored at 4°C

• Long-term storage: Aliquot and store at -20°C or -70°C

• Avoid repeated freeze-thaw cycles as this can significantly reduce enzymatic activity

• Store in buffer containing stabilizing agents (typical buffer: 20mM Tris-HCl buffer at pH 8.0 containing 0.15M NaCl, 10% glycerol, 1mM DTT)

A stability study comparing activity retention under different storage conditions is recommended before beginning extensive research with the enzyme.

What expression systems are most effective for producing recombinant human GDA with histidine tag?

Multiple expression systems have been employed for producing recombinant human GDA, each with distinct advantages depending on research requirements:

For most basic enzymatic studies, E. coli remains the preferred system due to cost-effectiveness and high yield. Conventional chromatography techniques following initial affinity purification using the histidine tag provide protein with >90% purity as determined by SDS-PAGE .

What purification strategy yields the highest purity recombinant human GDA?

A multi-step purification strategy is recommended for obtaining high-purity recombinant human GDA with histidine tag:

• Initial capture: Immobilized metal affinity chromatography (IMAC) utilizing the histidine tag's affinity for nickel or cobalt resin

• Intermediate purification: Ion exchange chromatography to separate based on charge differences

• Polishing step: Size exclusion chromatography to remove aggregates and achieve final purity

This approach consistently yields protein with >95% purity as determined by SDS-PAGE . The purified protein should be analyzed for both purity and specific activity, as high purity does not always correlate with optimal enzymatic function.

How can researchers address common challenges in recombinant human GDA expression?

When expressing recombinant human GDA, researchers frequently encounter challenges that can be addressed through methodological adjustments:

Challenge 1: Inclusion body formation in E. coli

• Reduce expression temperature to 16-18°C

• Use bacterial strains designed for improved folding (e.g., Rosetta, Origami)

• Co-express with molecular chaperones

• Optimize induction conditions (lower IPTG concentration)

Challenge 2: Low enzymatic activity despite high purity

• Verify protein folding using circular dichroism

• Ensure proper buffer conditions during purification and storage

• Add stabilizing agents (glycerol, reducing agents)

• Consider mammalian expression systems for proper post-translational modifications

Challenge 3: Protein aggregation during concentration/storage

• Add mild detergents below critical micelle concentration

• Maintain protein at concentrations below 1 mg/mL during processing

• Use buffer screening to identify optimal stability conditions

• Consider adding competitive inhibitors during storage

What are the established assays for measuring human GDA activity?

Several methodologies exist for measuring human GDA activity, each with specific advantages for particular research questions:

The recently developed real-time monitoring assay utilizing the isothiazolo guanine surrogate (tzGN) provides a significant advancement, allowing continuous monitoring of reaction kinetics through the spectroscopic differences between substrate and product .

How can researchers effectively study GDA inhibition?

For inhibitor studies with human GDA, a systematic approach is recommended:

• Initial screening: Use the fluorescence-based assay with tzGN to rapidly identify potential inhibitors by monitoring reduction in deamination rate .

• Inhibition characterization: Determine inhibition type and constants (Ki) through kinetic analysis using varying substrate and inhibitor concentrations.

• Validation methodology:

  • Confirm inhibition with natural substrate (guanine)

  • Verify specificity by testing against related deaminases

  • Assess cellular activity using cell-based assays

  • Evaluate reversibility through dilution or dialysis experiments

A pilot study demonstrated this approach with previously reported and novel inhibitors of GDA activity . When reporting inhibition results, researchers should clearly specify experimental conditions including enzyme concentration, substrate type and concentration, buffer composition, and temperature.

What controls are essential when studying GDA function in vitro?

When designing experiments to study GDA function in vitro, the following controls are essential:

Positive controls:

• Commercial GDA with verified activity

• Known substrate with established kinetic parameters

• Reaction conditions previously validated in literature

Negative controls:

• Heat-inactivated enzyme (95°C for 10 minutes)

• Reaction mixture without enzyme

• Reaction mixture without substrate

• Buffer-only control

Specificity controls:

• Related deaminases (e.g., adenosine deaminase)

• Non-substrate purines

• Catalytically inactive GDA mutant (if available)

All experiments should include appropriate blanks to account for background absorbance or fluorescence, particularly important when using the spectroscopic methods with surrogate substrates like tzGN .

How does human GDA interact with the microtubule network?

Beyond its canonical role in guanine metabolism, GDA (also known as CYPIN) functions as a cytoplasmic PSD95 interactor involved in microtubule assembly . Research approaches to investigate this non-metabolic function include:

• Co-localization studies: Immunofluorescence microscopy using anti-GDA antibodies and microtubule markers to visualize spatial relationships in cells

• Protein-protein interaction mapping:

  • Co-immunoprecipitation with microtubule components

  • Proximity labeling methods (BioID, APEX)

  • Fluorescence resonance energy transfer (FRET) to detect direct interactions

• Functional studies:

  • GDA overexpression and knockdown effects on microtubule dynamics

  • Analysis of microtubule polymerization rates in the presence of purified GDA

  • Live cell imaging of microtubule behavior with fluorescently tagged GDA

When investigating these interactions, it's critical to distinguish between direct effects and indirect consequences of altered guanine metabolism, which may independently affect cellular processes.

What is known about post-translational modifications of human GDA?

Post-translational modifications (PTMs) of GDA remain relatively understudied but potentially important for regulating both enzymatic activity and non-catalytic functions. Research methodologies to explore GDA PTMs include:

• Identification approaches:

  • Mass spectrometry of purified native GDA from tissue samples

  • Phosphoproteomic analysis from cells under various conditions

  • Western blotting with modification-specific antibodies

• Functional impact assessment:

  • Site-directed mutagenesis of modified residues

  • In vitro modification using purified modifying enzymes

  • Activity comparisons between modified and unmodified proteins

• Regulatory mechanisms:

  • Temporal analysis of modifications during cell cycle or differentiation

  • Effects of signaling pathway modulators on GDA modification status

  • Identification of regulatory enzymes (kinases, acetylases, etc.)

When studying PTMs, researchers should consider how the recombinant expression system may affect modification patterns, potentially necessitating mammalian expression systems rather than bacterial systems for certain studies.

How do different isoforms of human GDA differ in catalytic properties?

Multiple transcript variants encoding different isoforms have been identified for the human GDA gene . Systematic characterization of these isoforms includes:

• Expression profiling:

  • Tissue-specific expression patterns of each isoform

  • Developmental regulation of isoform expression

  • Subcellular localization differences

• Biochemical comparisons:

  • Enzyme kinetics (Km, Vmax, kcat) with natural and surrogate substrates

  • Inhibitor sensitivity profiles

  • Stability and pH optima differences

• Structural analysis:

  • Homology modeling of isoform-specific regions

  • Circular dichroism to assess secondary structure differences

  • Limited proteolysis to identify flexible or exposed regions

Each isoform should be recombinantly expressed with identical tags and under similar conditions to enable direct comparative analysis without introducing system-specific artifacts.

How can advanced spectroscopic methods enhance GDA research?

Recent developments in spectroscopic methods offer new approaches for studying GDA structure and function:

The development of emissive heterocyclic cores as GDA substrates represents a significant advancement in real-time monitoring capabilities. Unlike the thieno analog (thGN), the isothiazolo guanine surrogate (tzGN) undergoes effective enzymatic deamination by GDA and yields the spectroscopically distinct xanthine analog (tzX) . This spectral difference enables continuous monitoring of the enzymatic reaction, providing advantages over traditional discontinuous assays.

Future applications of this technology could include:

• High-throughput screening of GDA inhibitors

• Structure-activity relationship studies using modified surrogate substrates

• In cellulo GDA activity measurements using cell-permeable fluorescent substrates

• Binding studies using fluorescence anisotropy or quenching mechanisms

What approaches can identify novel GDA inhibitors for research applications?

Systematic approaches for identifying novel GDA inhibitors include:

• High-throughput screening methodology:

  • Primary screen using the fluorescence-based tzGN deamination assay

  • Secondary validation with natural substrate

  • Counter-screening against related deaminases for selectivity

  • Cellular activity confirmation in GDA-expressing cells

• Structure-based design strategies:

  • In silico docking using homology models or crystal structures

  • Fragment-based screening targeting the active site

  • Transition-state analog design based on reaction mechanism

  • Allosteric inhibitor development targeting non-catalytic regions

• Rational expansion from known inhibitors:

  • Modification of previously reported inhibitors

  • Scaffold hopping from inhibitors of related deaminases

  • Natural product derivative screening

A small pilot study has already demonstrated the utility of real-time monitoring for inhibitor evaluation, including one previously reported inhibitor and two new inhibitors , providing a foundation for expanded screening campaigns.

How can researchers investigate GDA's role in specific disease contexts?

To investigate GDA's potential role in disease contexts, researchers can employ these methodological approaches:

• Clinical correlation studies:

  • GDA activity measurements in patient samples

  • Genetic association studies of GDA variants with disease outcomes

  • Expression analysis in disease tissues versus controls

• Mechanistic investigations:

  • Disease model systems with GDA modulation (overexpression/knockdown)

  • Metabolomic profiling to identify downstream effects of altered GDA activity

  • Rescue experiments to establish causality in phenotypic changes

• Therapeutic targeting assessment:

  • Effects of specific inhibitors on disease phenotypes

  • Combination approaches targeting multiple nodes in purine metabolism

  • Biomarker development for patient stratification

When designing these studies, researchers should consider the dual functions of GDA in both metabolism and cytoskeletal regulation, as disease phenotypes could result from disruption of either or both functions.