The ability of one enzyme to catalyse multiple, mechanistically distinct transformations likely played a crucial role in organisms’ abilities to adapt to changing external stimuli in the past and can still be observed in extant enzymes. Given the importance of catalytic promiscuity in nature, enzyme designers have recently begun to create catalytically promiscuous enzymes in order to expand the canon of transformations catalysed by proteins. This article aims to both critically review different strategies for the design of enzymes that display catalytic promiscuity for new-to-nature reactions and highlight the successes of subsequent directed-evolution efforts to fine-tune these novel reactivities. For the former, we put a particular emphasis on the creation, stabilization and repurposing of reaction intermediates, which are key for unlocking new activities in an existing or designed active site. For the directed evolution of the resulting catalysts, we contrast approaches for enzyme design that make use of components found in nature and those that achieve new reactivities by incorporating synthetic components. Following the critical analysis of selected examples that are now available, we close this Review by providing a set of considerations and design principles for enzyme engineers, which will guide the future generation of efficient artificial enzymes for synthetically useful, abiotic transformations.

过去，一种酶催化多种机械上不同的转化的能力可能在生物适应外界刺激变化的能力中起着至关重要的作用，并且仍然可以在现存的酶中观察到。考虑到自然界中催化混杂的重要性，酶设计者最近开始创建催化混杂酶，以扩展蛋白质催化的转化标准。本文旨在严格审查酶设计的不同策略，这些策略显示对新自然反应的催化混杂，并着重介绍了随后的定向进化方法的成功，以微调这些新的反应性。对于前者，我们特别强调反应中间体的产生，稳定化和再利用，这是在现有或设计的活动站点中解锁新活动的关键。对于所得催化剂的定向进化，我们对比了酶设计方法，这些方法利用了自然界中发现的组分以及通过掺入合成组分而实现新反应性的酶。在对现在可用的精选实例进行严格分析之后，我们通过为酶工程师提供一系列考虑因素和设计原则来结束本综述，这将指导未来一代可用于合成有用的非生物转化的高效人工酶的开发。我们对比了利用自然界中发现的成分和通过掺入合成成分而获得新反应性的酶设计方法。在对现在可用的精选实例进行严格分析之后，我们通过为酶工程师提供一系列考虑因素和设计原则来结束本综述，这将指导未来一代可用于合成有用的非生物转化的高效人工酶的开发。我们对比了利用自然界中发现的成分和通过掺入合成成分而获得新反应性的酶设计方法。在对现在可用的精选实例进行严格分析之后，我们通过为酶工程师提供一系列考虑因素和设计原则来结束本综述，这将指导未来一代可用于合成有用的非生物转化的高效人工酶的开发。