Recombinant Dictyostelium discoideum GMP synthase [glutamine-hydrolyzing] (guaA), partial

Molecular and Functional Characteristics

GMP synthase (guaA) catalyzes the final step in guanine monophosphate (GMP) biosynthesis, converting xanthosine monophosphate (XMP) to GMP using glutamine as a nitrogen donor. The Dictyostelium enzyme is classified as a Class I glutamine amidotransferase, characterized by a conserved catalytic triad (Cys-His-Glu) for glutamine hydrolysis .

Key enzymatic activities:

• ATP-dependent amination: Transfers an amino group from glutamine to XMP.

• Glutamine hydrolysis: Mediated by the N-terminal glutaminase domain .

The term "partial" indicates that the recombinant form lacks specific domains, likely engineered to simplify purification or study isolated functional regions.

Recombinant Expression in Dictyostelium discoideum

Dictyostelium is a favored eukaryotic host for recombinant protein production due to its ability to perform post-translational modifications akin to higher eukaryotes . Key advantages include:

• High-throughput compatibility: Facilitates large-scale screening .

• Endogenous promoters: Enable precise regulation of gene expression .

Expression workflow:

1. Cloning of partial guaA into plasmid vectors under constitutive promoters.

2. Transformation into Dictyostelium AX2 or AX3 strains.

3. Purification via affinity tags (e.g., His-tag) .

Functional and Genetic Studies

In Dictyostelium, guaA is critical for nucleotide metabolism during developmental stages. Knockout studies in related bacteria (e.g., Borrelia burgdorferi) demonstrate that guaA disruption abolishes infectivity, underscoring its essential role . In Dictyostelium, RNA interference (RNAi) screens reveal that gene silencing mechanisms target guaA homologs, though residual activity persists during development .

Key findings:

• Enzymatic activity: Recombinant guaA retains kinetic properties comparable to native enzymes (e.g., Kₘ for glutamine ≈ 0.5 mM) .

• Regulatory interactions: Associates with IMP dehydrogenase (guaB) in purine salvage pathways .

Applications and Research Implications

1. Drug discovery: Target for antiparasitic agents due to conserved catalytic sites .

2. Metabolic engineering: Optimization of nucleotide biosynthesis in synthetic biology .

3. Structural biology: Partial constructs enable crystallography of isolated domains .

Challenges and Future Directions

• Domain truncation effects: Partial constructs may lack allosteric regulation sites.

• Heterologous expression: Solubility and folding issues in bacterial systems necessitate eukaryotic hosts like Dictyostelium .

Future studies should focus on full-length guaA characterization and leveraging Dictyostelium’s genetic tools for high-resolution functional mapping .