Mechanism-Based Post-Translational Modification and Inactivation in Terpene Synthases

Terpenes are ubiquitous natural chemicals with diverse biological functions spanning all three domains of life. In specialized metabolism, the active sites of terpene synthases (TPSs) evolve in shape and reactivity to direct the biosynthesis of a myriad of chemotypes for organismal fitness. As most terpene biosynthesis mechanistically involves highly reactive carbocationic intermediates, the protein surfaces catalyzing these cascade reactions possess reactive regions possibly prone to premature carbocation capture and potentially enzyme inactivation. Here, we show using proteomic and X-ray crystallographic analyses that cationic intermediates undergo capture by conserved active site residues leading to inhibitory self-alkylation. Moreover, the level of cation-mediated inactivation increases with mutation of the active site, upon changes in the size and structure of isoprenoid diphosphate substrates, and alongside increases in reaction temperatures. TPSs that individually synthesize multiple products are less prone to self-alkylation then TPSs possessing relatively high product specificity. In total, the results presented suggest that mechanism-based alkylation represents an overlooked mechanistic pressure during the evolution of cation-derived terpene biosynthesis.萜类化合物(Terpenes)是一类广泛存在的天然化学物质，具备覆盖生命三大域的多样生物学功能。在特化代谢过程中，萜合酶(TPSs)的活性位点会在构象与反应活性层面发生演化，以催化合成海量各异化学型的萜类产物，提升机体适应性。由于多数萜类生物合成都涉及高反应活性的碳正离子中间体，催化这类级联反应的蛋白质表面存在易发生过早碳正离子捕获的反应性区域，进而可能引发酶失活。本研究通过蛋白质组学与X射线晶体学分析发现，阳离子中间体可被保守的活性位点残基捕获，进而引发具有抑制作用的自身烷基化反应。此外，阳离子介导的酶失活程度会随活性位点突变、异戊二烯二磷酸底物的尺寸与结构改变，以及反应温度升高而加剧。相较于产物特异性较高的萜合酶，可同时合成多种产物的萜合酶更不易发生自身烷基化。综上，本研究结果表明，基于反应机制的烷基化作用，是碳正离子依赖型萜类生物合成演化过程中一种被长期忽视的机制性选择压力。