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 , 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 PKS2, TR, suggests differences 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 TR and observed that it is capable of 4 electron (4e) reduction of octanoyl-CoA to the primary alcohol, octanol, utilizing NADH. 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 TR using a fluorescence assay and determined the TR binding affinity for cofactor and substrates using isothermal titration calorimetry (ITC). We additionally show that TR is capable of reducing an acyl carrier protein (ACP)-tethered substrate by liquid chromatography mass spectrometry and determine that TR binds to holo-ACP4, its predicted cognate ACP, with a of 5.75 ± 0.77 μM. Finally, our transcriptional analysis shows that PKS2 is upregulated ∼4-fold in the parasite's cyst-forming bradyzoite stage compared to tachyzoites. Our study identifies features that distinguish PKS2 from well-characterized systems in bacteria and fungi and suggests it aids the cyst stage.