Foran Mia E, Auckloo Nazia B, Ho Y T Candace, Liu Shaonan, Hai Yang, Jenner Matthew
Department of Chemistry, University of Warwick Coventry CV4 7AL UK
Department of Chemistry and Biochemistry, University of California Santa Barbara 93110 Santa Barbara CA USA.
Chem Sci. 2025 Jun 23. doi: 10.1039/d5sc01027f.
Fungal highly reducing polyketide synthases (hrPKSs) are remarkable multidomain enzymes that catalyse the biosynthesis of a diverse range of structurally complex compounds. During biosynthesis, the ketosynthase (KS) and acyltransferase (AT) domains of the condensing region are visited by the acyl carrier protein (ACP) domain during every cycle, catalysing chain priming and elongation reactions. Despite their significance, our comprehension of how these steps contribute to biosynthetic fidelity remains poorly understood. Using the SimG hrPKS from the cyclosporin pathway as a model system, we report the full reconstitution of each catalytic step carried out by a hrPKS condensing region. Application of intact protein mass spectrometry shows that the AT domain operates selective acyl transfer to the ACP domain, ensuring precise loading of the coenzyme A (CoA)-tethered acetyl and malonyl starter/extender units, which is governed by a substrate recycling mechanism and kinetic parameters towards each substrate. The KS domain is shown to select for the correct starter unit for transacylation and subsequent chain elongation, and was also able to utilise methylmalonyl-CoA. This work provides the first comprehensive interrogation of a fungal hrPKS condensing region, which both affirms and expands our understanding of how these systems function, and provides a framework for future bioengineering efforts.
真菌高度还原型聚酮合酶（hrPKSs）是一类非凡的多结构域酶，可催化多种结构复杂化合物的生物合成。在生物合成过程中，缩合区域的酮合成酶（KS）和酰基转移酶（AT）结构域在每个循环中都会与酰基载体蛋白（ACP）结构域相互作用，催化链引发和延伸反应。尽管它们很重要，但我们对这些步骤如何促进生物合成保真度的理解仍然很差。我们以环孢素途径中的SimG hrPKS作为模型系统，报告了hrPKS缩合区域执行的每个催化步骤的完全重构。完整蛋白质质谱分析表明，AT结构域对ACP结构域进行选择性酰基转移，确保辅酶A（CoA）连接的乙酰基和丙二酰起始/延伸单元的精确加载，这受底物循环机制和对每种底物的动力学参数控制。KS结构域被证明能选择正确的起始单元进行转酰化和随后的链延伸，并且还能够利用甲基丙二酰-CoA。这项工作首次对真菌hrPKS缩合区域进行了全面研究，既肯定并扩展了我们对这些系统功能的理解，也为未来的生物工程工作提供了框架。
