Aromatic prenyltransferases (aPTases) transfer prenyl moieties from isoprenoid donors to various aromatic acceptors, some of which have the rare property of extreme enzymatic promiscuity toward both a variety of prenyl donors and a large diversity of acceptors. In this study, we discovered a new aPTase, AtaPT, from Aspergillus terreus that exhibits unprecedented promiscuity toward diverse aromatic acceptors and prenyl donors and also yields products with a range of prenylation patterns. Systematic crystallographic studies revealed various discrete conformations for ligand binding with donor-dependent acceptor specificity and multiple binding sites within a spacious hydrophobic substrate-binding pocket. Further structure-guided mutagenesis of active sites at the substrate-binding pocket is responsible for altering the specificity and promiscuity toward substrates and the diversity of product prenylations. Our study reveals the molecular mechanism underlying the promiscuity of AtaPT and suggests an efficient protein engineering strategy to generate new prenylated derivatives in drug discovery applications.

芳香异戊二烯基转移酶（aPTase）将异戊二烯供体中的异戊二烯基团转移到各种芳族受体上，其中一些对多种异戊二烯基供体和多种受体都具有极强的酶促混杂特性。在这项研究中，我们从土曲霉中发现了一种新的aPTase，AtaPT对各种芳香族受体和异戊二烯供体表现出前所未有的混杂性，并且还可以产生具有各种异戊二烯化模式的产品。系统的晶体学研究揭示了具有供体依赖性受体特异性的配体结合的各种离散构象以及在宽敞的疏水性底物结合口袋中的多个结合位点。在底物结合口袋处的活性位点的进一步结构引导的诱变负责改变对底物的特异性和混杂性以及产物烯丙基化的多样性。我们的研究揭示了AtaPT混杂的分子机制，并提出了一种有效的蛋白质工程策略，可在药物开发应用中产生新的异戊二烯衍生物。