Exploring the Chain Release Mechanism from an Atypical Apicomplexan Polyketide Synthase

Abstract

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 parasiteToxoplasma gondii, TgPKS2, which contains a unique putative chain release mechanism that includes ketosynthase (KS) and thioester reductase (TR) domains. Our bioinformatic analysis of the thioester reductase ofTgPKS2,TgTR, 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 isolatedTgTR and observed that it is capable of 4 electron (4e-) reduction of octanoyl-CoA to the primary alcohol, octanol, utilizing NADH as a cofactor.TgTR 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 ofTgTR using a fluorescence assay and determined theTgTR binding affinity for cofactor and substrates using isothermal titration calorimetry (ITC). We additionally show thatTgTR is capable of reducing an acyl carrier protein (ACP)-tethered substrate by liquid chromatography mass spectrometry and determine thatTgTR binds to holo-TgACP4, its predicted cognate ACP, with aKDof 5.75 ± 0.77 µM. Finally, our transcriptional analysis shows thatTgPKS2 is upregulated ∼4-fold in the parasite’s cyst-forming bradyzoite stage compared to tachyzoites. Our study identifies features that distinguishTgPKS2 from well-characterized systems in bacteria and fungi, and suggests it aids theT. gondiicyst stage. Together, this work increases our knowledge of PKS thioester reductase domains and advances our understanding of unconventional polyketide chain termination mechanisms.