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Exploring the Chain Release Mechanism from an Atypical Apicomplexan Polyketide Synthase.
Aaron M Keeler1, Porter E Petruzziello1, Elizabeth G Boger1
1Department of Chemistry, Duke University, Durham, North Carolina 27708, United States.
Biochemistry
|August 9, 2023
Summary
Toxoplasma gondii thioester reductase (TgTR) reduces octanoyl-CoA to octanol using NADH, distinct from other systems. This enzyme is upregulated in the parasite's cyst stage, suggesting a role in Toxoplasma gondii development.
Area of Science:
- Biochemistry
- Molecular Biology
- Parasitology
Background:
- Polyketide synthases (PKSs) are crucial enzymes for producing diverse polyketides across life.
- The apicomplexan parasite *Toxoplasma gondii* possesses a unique type I PKS, *Tg*PKS2, with a distinct chain release mechanism involving ketosynthase (KS) and thioester reductase (TR) domains.
- Bioinformatic analysis suggested *Tg*TR differs from known systems and may represent a conserved apicomplexan release mechanism.
Purpose of the Study:
- To biochemically characterize the *Toxoplasma gondii* thioester reductase (*Tg*TR) and its role in the *Tg*PKS2 pathway.
- To investigate the substrate specificity and cofactor requirements of *Tg*TR.
- To determine the interaction between *Tg*TR and its cognate acyl carrier protein (ACP).
Main Methods:
- Purification and biochemical assays of isolated *Tg*TR, including substrate reduction and cofactor dependency.
- Enzyme kinetics and binding affinity studies using fluorescence assays and isothermal titration calorimetry (ITC).
- Analysis of *Tg*TR activity on ACP-tethered substrates via liquid chromatography-mass spectrometry (LC-MS).
- Transcriptional analysis of *Tg*PKS2 expression in different *T. gondii* stages.
Main Results:
- *Tg*TR catalyzes the 4-electron reduction of octanoyl-CoA to octanol, specifically utilizing NADH and not NADPH.
- *Tg*TR demonstrates catalytic efficiency and binds its cognate holo-*Tg*ACP4 with a dissociation constant (*K*D) of 5.75 ± 0.77 μM.
- The *Tg*PKS2 gene is significantly upregulated (approximately 4-fold) in the bradyzoite stage of *T. gondii* compared to the tachyzoite stage.
Conclusions:
- *Tg*TR possesses unique biochemical properties distinguishing it from bacterial and fungal PKS thioester reductases.
- The findings suggest a conserved apicomplexan chain release mechanism within *T. gondii* PKS.
- *Tg*PKS2 and its *Tg*TR component likely play a role in the parasite's cyst-forming bradyzoite stage.
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