Fage Christopher D, Passmore Munro, Tatman Ben P, Smith Helen G, Jian Xinyun, Dissanayake Upeksha C, Foran Mia E, Cisneros G Andrés, Challis Gregory L, Lewandowski Józef R, Jenner Matthew
Department of Chemistry, University of Warwick, Coventry CV4 7AL, U.K.
Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Saclay, 91198 Gif-sur-Yvette, France.
JACS Au. 2024 Nov 18;5(1):144-157. doi: 10.1021/jacsau.4c00837. eCollection 2025 Jan 27.
Polyketide synthases (PKSs) are multidomain enzymatic assembly lines that biosynthesize a wide selection of bioactive natural products from simple building blocks. In contrast to their -acyltransferase (AT) counterparts, -AT PKSs rely on stand-alone ATs to load extender units onto acyl carrier protein (ACP) domains embedded in the core PKS machinery. -AT PKS gene clusters also encode stand-alone acyl hydrolases (AHs), which are predicted to share the overall fold of ATs but function like type II thioesterases (TEs), hydrolyzing aberrant acyl chains from ACP domains to promote biosynthetic efficiency. How AHs specifically target short acyl chains, in particular acetyl groups, tethered as thioesters to the substrate-shuttling ACP domains, with hydrolytic rather than acyl transfer activity, has remained unclear. To answer these questions, we solved the first structure of an AH and performed structure-guided activity assays on active site variants. Our results offer key insights into chain length control and selection against coenzyme A-tethered substrates, and clarify how the interaction interface between AHs and ACP domains contributes to recognition of cognate and noncognate ACP domains. Combining our experimental findings with molecular dynamics simulations allowed for the construction of a data-driven model of an AH:ACP domain complex. Our results advance the currently incomplete understanding of polyketide biosynthesis by -AT PKSs, and provide foundations for future bioengineering efforts to offload biosynthetic intermediates or enhance product yields.
聚酮合酶（PKSs）是多结构域酶促装配线，可从简单的构件生物合成多种生物活性天然产物。与其β-酰基转移酶（AT）对应物不同，α-AT PKSs依赖独立的AT将延伸单元加载到嵌入核心PKS机制中的酰基载体蛋白（ACP）结构域上。α-AT PKS基因簇还编码独立的酰基水解酶（AHs），预计其与AT具有相同的整体折叠，但功能类似于II型硫酯酶（TEs），从ACP结构域水解异常的酰基链以提高生物合成效率。AHs如何特异性靶向以硫酯形式连接到底物穿梭ACP结构域上的短酰基链，尤其是乙酰基，且具有水解而非酰基转移活性，仍不清楚。为了回答这些问题，我们解析了AH的首个结构，并对活性位点变体进行了结构导向的活性测定。我们的结果为链长控制和针对辅酶A连接底物的选择提供了关键见解，并阐明了AH与ACP结构域之间的相互作用界面如何有助于识别同源和非同源ACP结构域。将我们的实验结果与分子动力学模拟相结合，构建了一个数据驱动的AH：ACP结构域复合物模型。我们的结果推进了目前对α-AT PKSs聚酮生物合成尚不完整的理解，并为未来卸载生物合成中间体或提高产物产量的生物工程努力奠定了基础。
