Exploring the Chain Release Mechanism from an Atypical Apicomplexan Polyketide Synthase

Polyketide synthases (PKSs) are megaenzymes that form chemically diverse polyketides and are found within the genomes of nearly all classes of life. We recently discovered the type I PKS from the apicomplexan parasite Toxoplasma gondii, Tg PKS2, which contains a unique putative chain release mechanism that includes ketosynthase (KS) and thioester reductase (TR) domains. Our bioinformatic analysis of the thioester reductase of Tg PKS2, Tg TR, suggests differences in putative apicomplexan reductase domains compared to other systems and hints at a possibly conserved release mechanism within the apicomplexan subclass Coccidia. To evaluate this release module, we first isolated Tg TR and observed that it is capable of 4 electron (4e ^- ) reduction of octanoyl-CoA to the primary alcohol, octanol, utilizing NADH as a cofactor. Tg TR was also capable of generating octanol in the presence of octanal and NADH, but no reactions were observed when NADPH was supplied as a cofactor. To biochemically characterize the protein, we measured the catalytic efficiency of Tg TR using a fluorescence assay and determined the Tg TR binding affinity for cofactor and substrates using isothermal titration calorimetry (ITC). We additionally show that Tg TR is capable of reducing an acyl carrier protein (ACP)-tethered substrate by liquid chromatography mass spectrometry and determine that Tg TR binds to holo- Tg ACP4, its predicted cognate ACP, with a K _D of 5.75 ± 0.77 µM. Finally, our transcriptional analysis shows that Tg PKS2 is upregulated ∼4-fold in the parasite’s cyst-forming bradyzoite stage compared to tachyzoites. Our study identifies features that distinguish Tg PKS2 from well-characterized systems in bacteria and fungi, and suggests it aids the T. gondii cyst stage. Together, this work increases our knowledge of PKS thioester reductase domains and advances our understanding of unconventional polyketide chain termination mechanisms.