Münch Judith, Soler Jordi, Gildor-Cristal Ofir, Fleishman Sarel J, Garcia-Borràs Marc, Weissenborn Martin J
Institute of Chemistry, Martin Luther-University Halle-Wittenberg, Weinbergweg 22, Halle (Saale) 06120, Germany.
Institut de Qui'mica Computacional i Catàlisi and Departament de Qui'mica, Universitat de Girona, Carrer Maria Aurèlia Capmany 69, Girona 17003 Catalonia, Spain.
ACS Catal. 2025 Jul 14;15(15):12741-12755. doi: 10.1021/acscatal.5c02412. eCollection 2025 Aug 1.
The selective oxyfunctionalization of terpenes remains a major challenge in chemical synthesis and is of significant industrial importance. This study presents a computational enzyme design approach based on an AlphaFold2 model of an unspecific peroxygenase (UPO). Using the FuncLib algorithm, only 50 variants were required, and they exhibit remarkable advancements. All 50 designs retained 100% measurable activity across the tested substrate panel, with each design showing activity on at least one substrate. Among the terpene substrates, improvements in activity varied considerably: while some substrates had only a single design exhibiting a ≥2-fold increase in activity, the top-performing substrate had 26 such designs. The most active design per terpene substrate showed enhancements ranging from 2.2-fold to 7.1-fold relative to the wild type. In addition to increased activity, many designs also demonstrated useful and dramatic shifts in regio-, chemo-, and stereoselectivity. Regioselectivity for the energetically less favored 3-hydroxy-β-damascone increased from 3 to 46%. Particularly striking is the dramatic improvement in chemoselectivity for the oxidation of geraniol and nerol to citral A (>99%) and citral B (89%), respectively. While wild-type UPO exhibited only a moderate selectivity of 40% for citral A and 72% for citral B, our computationally designed variants displayed significantly enhanced product preference and up to a 4.5-fold increase in activity. Additionally, further products not found with the wild-type enzyme, such as isopiperitenol from limonene and epoxides from geraniol and nerol, were synthesized. For the hydroxylation of β-ionone, the enantioselectivity was inverted to a ratio of 1:99 from ()- to ()-4-hydroxy-β-ionone. FuncLib-enabled active-site remodeling allowed us to generate a small yet highly diverse enzyme panel that significantly outperformed the wild type across multiple synthetic challenges. The best-performing variants, such as design 4 and design 11 (both 4 mutations), exhibit improvements that result from epistatic effects. MD simulations demonstrated that these mutations collectively reshape the active site, allowing for regio- and chemoselectivities that are difficult to achieve by single-point mutations. Herein, we demonstrate the potential of in silico-guided approaches to rapidly develop highly selective biocatalysts for synthetic applications.
萜类化合物的选择性氧官能化仍然是化学合成中的一项重大挑战，并且具有重要的工业意义。本研究提出了一种基于非特异性过氧合酶（UPO）的AlphaFold2模型的计算酶设计方法。使用FuncLib算法，仅需50个变体，且它们展现出显著进展。所有50种设计在测试的底物组中均保留了100%可测量的活性，每种设计在至少一种底物上表现出活性。在萜类底物中，活性的提高差异很大：虽然有些底物只有一种设计的活性提高了≥2倍，但表现最佳的底物有26种这样的设计。每种萜类底物最具活性的设计相对于野生型显示出2.2倍至7.1倍的增强。除了活性增加外，许多设计还在区域选择性、化学选择性和立体选择性方面表现出有用且显著的变化。对能量上较不利的3-羟基-β-大马酮的区域选择性从3%提高到了46%。特别引人注目的是，香叶醇和橙花醇分别氧化为柠檬醛A（>99%）和柠檬醛B（89%）的化学选择性有了显著提高。虽然野生型UPO对柠檬醛A的选择性仅为中等的40%，对柠檬醛B为72%，但我们通过计算设计的变体显示出显著增强的产物偏好，活性提高了4.5倍。此外，还合成了野生型酶未发现的其他产物，如来自柠檬烯的异胡椒酮醇以及来自香叶醇和橙花醇的环氧化物。对于β-紫罗兰酮的羟基化，对映选择性从（）-4-羟基-β-紫罗兰酮反转到了（）-4-羟基-β-紫罗兰酮，比例为1:99。基于FuncLib的活性位点重塑使我们能够生成一个小而高度多样的酶组，在多个合成挑战中显著优于野生型。表现最佳的变体，如设计4和设计11（均为4个突变），表现出的改进是由上位效应导致的。分子动力学模拟表明，这些突变共同重塑了活性位点，实现了单点突变难以达到的区域选择性和化学选择性。在此，我们展示了计算机辅助方法在快速开发用于合成应用的高选择性生物催化剂方面的潜力。
