Keeler Aaron M, D'Ambrosio Hannah K, Ganley Jack G, Derbyshire Emily R
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.
ACS Chem Biol. 2023 Apr 21;18(4):785-793. doi: 10.1021/acschembio.2c00783. Epub 2023 Mar 9.
Natural products play critical roles as antibiotics, anticancer therapeutics, and biofuels. Polyketides are a distinct natural product class of structurally diverse secondary metabolites that are synthesized by polyketide synthases (PKSs). The biosynthetic gene clusters that encode PKSs have been found across nearly all realms of life, but those from eukaryotic organisms are relatively understudied. A type I PKS from the eukaryotic apicomplexan parasite ,PKS2, was recently discovered through genome mining, and the functional acyltransferase (AT) domains were found to be selective for malonyl-CoA substrates. To further characterize PKS2, we resolved assembly gaps within the gene cluster, which confirmed that the encoded protein consists of three distinct modules. We subsequently isolated and biochemically characterized the four acyl carrier protein (ACP) domains within this megaenzyme. We observed self-acylation─or substrate acylation without an AT domain─for three of the four PKS2 ACP domains with CoA substrates. Furthermore, CoA substrate specificity and kinetic parameters were determined for all four unique ACPs. ACP2-4 were active with a wide scope of CoA substrates, while ACP1 from the loading module was found to be inactive for self-acylation. Previously, self-acylation has only been observed in type II systems, which are enzymes that act with one another, and this represents the first report of this activity in a modular type I PKS whose domains function . Site-directed mutagenesis of specific PKS2 ACP3 acidic residues near the phosphopantetheinyl arm demonstrated that they influence self-acylation activity and substrate specificity, possibly by influencing substrate coordination or phosphopantetheinyl arm activation. Further, the lack of PKS2 ACP self-acylation with acetoacetyl-CoA, which is utilized by previously characterized type II PKS systems, suggests that the substrate carboxyl group may be critical for PKS2 ACP self-acylation. The unexpected properties observed from PKS ACP domains highlight their distinction from well-characterized microbial and fungal systems. This work expands our understanding of ACP self-acylation beyond type II systems and helps pave the way for future studies on biosynthetic enzymes from eukaryotes.
天然产物在抗生素、抗癌治疗药物和生物燃料方面发挥着关键作用。聚酮化合物是一类独特的天然产物，属于结构多样的次生代谢产物，由聚酮合酶（PKSs）合成。编码PKSs的生物合成基因簇几乎在生命的所有领域都有发现，但来自真核生物的那些基因簇相对研究较少。最近通过基因组挖掘发现了一种来自真核顶复门寄生虫的I型PKS，即PKS2，并且发现其功能性酰基转移酶（AT）结构域对丙二酰辅酶A底物具有选择性。为了进一步表征PKS2，我们解决了基因簇内的组装缺口，这证实了编码的蛋白质由三个不同的模块组成。我们随后分离并对这种巨型酶中的四个酰基载体蛋白（ACP）结构域进行了生化表征。我们观察到四个PKS2 ACP结构域中的三个与辅酶A底物发生了自我酰化——即没有AT结构域的底物酰化。此外，还测定了所有四个独特ACP的辅酶A底物特异性和动力学参数。ACP2 - 4对多种辅酶A底物具有活性，而来自装载模块的ACP1被发现对自我酰化无活性。以前，自我酰化仅在II型系统中被观察到，II型系统是相互作用的酶，而这是这种活性在其结构域起作用的模块化I型PKS中的首次报道。对PKS2 ACP3靠近磷酸泛酰巯基乙胺臂的特定酸性残基进行定点诱变表明，它们可能通过影响底物配位或磷酸泛酰巯基乙胺臂的激活来影响自我酰化活性和底物特异性。此外，PKS2 ACP与乙酰乙酰辅酶A不发生自我酰化，而之前表征的II型PKS系统利用乙酰乙酰辅酶A，这表明底物羧基可能对PKS2 ACP自我酰化至关重要。从PKS ACP结构域观察到的意外特性突出了它们与已充分表征的微生物和真菌系统的差异。这项工作扩展了我们对ACP自我酰化的理解，超越了II型系统，并有助于为未来对真核生物生物合成酶的研究铺平道路。
