Department of Chemistry, Duke University, Durham, North Carolina 27708, United States.
Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710, United States.
Biochemistry. 2023 Sep 5;62(17):2677-2688. doi: 10.1021/acs.biochem.3c00272. Epub 2023 Aug 9.
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.
聚酮合酶（PKSs）是形成化学多样的聚酮类化合物的巨型酶，存在于几乎所有生命形式的基因组中。我们最近发现了顶复门寄生虫中的 I 型 PKS，即 PKS2，它包含一种独特的假定链释放机制，其中包括酮合酶（KS）和硫酯还原酶（TR）结构域。我们对 PKS2 的硫酯还原酶 TR 的生物信息学分析表明，它与其他系统存在差异，并暗示在顶复门子类球虫中存在可能保守的释放机制。为了评估这个释放模块，我们首先分离了 TR，并观察到它能够利用 NADH 将辛酰基-CoA 还原为仲醇辛醇，完成 4 个电子（4e）的还原。TR 还能够在存在辛醛和 NADH 的情况下生成辛醇，但当 NADPH 作为辅助因子供应时，没有观察到反应。为了对该蛋白进行生化表征，我们使用荧光测定法测量了 TR 的催化效率，并使用等温滴定量热法（ITC）测定了 TR 对辅助因子和底物的结合亲和力。我们还证明，TR 能够通过液相色谱质谱法还原与酰基载体蛋白（ACP）相连的底物，并确定 TR 与预测的同源 ACP holo-ACP4 的结合亲和力为 5.75±0.77 μM。最后，我们的转录分析表明，与裂殖体（tachyzoites）相比，PKS2 在顶复门寄生虫的囊形成缓殖子（cyst-forming bradyzoites）阶段的转录水平上调了约 4 倍。我们的研究确定了 PKS2 与细菌和真菌中已充分研究的系统的区别特征，并表明它有助于囊阶段的形成。
