Facultad de Ciencias Bioquímicas y Farmacéuticas, Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET), Universidad Nacional de Rosario, Argentina.
Facultad de Ciencias Bioquímicas y Farmacéuticas, Instituto de Química de Rosario (IQUIR-CONICET), Universidad Nacional de Rosario, Argentina.
FEBS J. 2018 Dec;285(23):4494-4511. doi: 10.1111/febs.14675. Epub 2018 Oct 25.
Iterative type I polyketide synthases (PKS) are megaenzymes essential to the biosynthesis of an enormously diverse array of bioactive natural products. Each PKS contains minimally three functional domains, β-ketosynthase (KS), acyltransferase (AT), and acyl carrier protein (ACP), and a subset of reducing domains such as ketoreductase (KR), dehydratase (DH), and enoylreductase (ER). The substrate selection, condensation reactions, and β-keto processing of the polyketide growing chain are highly controlled in a programmed manner. However, the structural features and mechanistic rules that orchestrate the iterative cycles, processing domains functionality, and chain termination in this kind of megaenzymes are often poorly understood. Here, we present a biochemical and functional characterization of the KS and the AT domains of a PKS from the mallard duck Anas platyrhynchos (ApPKS). ApPKS belongs to an animal PKS family phylogenetically more related to bacterial PKS than to metazoan fatty acid synthases. Through the dissection of the ApPKS enzyme into mono- to didomain fragments and its reconstitution in vitro, we determined its substrate specificity toward different starters and extender units. ApPKS AT domain can effectively transfer acetyl-CoA and malonyl-CoA to the ApPKS ACP stand-alone domain. Furthermore, the KS and KR domains, in the presence of Escherichia coli ACP, acetyl-CoA, and malonyl-CoA, showed the ability to catalyze the chain elongation and the β-keto reduction steps necessary to yield a 3-hydroxybutyryl-ACP derivate. These results provide new insights into the catalytic efficiency and specificity of this uncharacterized family of PKSs.
迭代型 I 聚酮合酶（PKS）是生物合成极其多样化生物活性天然产物所必需的巨型酶。每个 PKS 至少包含三个功能域，β-酮基合酶（KS）、酰基转移酶（AT）和酰基载体蛋白（ACP），以及一些还原酶如酮还原酶（KR）、脱水酶（DH）和烯酰还原酶（ER）。聚酮生长链的底物选择、缩合反应和β-酮处理以程序化的方式得到高度控制。然而，这种巨型酶中迭代循环、加工域功能和链终止的结构特征和机制规则往往理解不足。在这里，我们对鸭（Anas platyrhynchos）的 PKS 的 KS 和 AT 结构域进行了生化和功能表征。ApPKS 属于动物 PKS 家族，在系统发育上与细菌 PKS 比与后生动物脂肪酸合酶更相关。通过将 ApPKS 酶剖分为单域至双域片段并在体外重建，我们确定了其对不同起始和扩展单元的底物特异性。ApPKS AT 结构域可以有效地将乙酰-CoA 和丙二酰-CoA 转移到 ApPKS ACP 独立结构域。此外，在存在大肠杆菌 ACP、乙酰-CoA 和丙二酰-CoA 的情况下，KS 和 KR 结构域表现出催化链延伸和β-酮还原步骤的能力，这是生成 3-羟基丁酰-ACP 衍生物所必需的。这些结果为这一未表征的 PKS 家族的催化效率和特异性提供了新的见解。
