Imine Reductases: Biochemistry, Engineering and Application

合成具有生物活性的药物和农用化合物需要以一种非常特殊的方式合成化学中间体，只给出两种可能的异构体中的一种，因为不想要的形式可能是不活跃的，甚至是有毒的。药物合成中一些最重要的化学中间体是胺，即构成知名药物(如β-受体阻滞剂)和止痛药(如布洛芬)的一部分的含氮化合物。以单一异构体的形式合成胺是一个巨大的挑战，越来越多的学者和工业家都转向酶--自然界的生物催化剂--作为制造这些化合物的一种有效方法。酶不仅自然具有生成单一异构体所需的选择性，而且从可持续、绿色化学的角度来看，它们也很有吸引力，因为它们在常温常压下工作，不需要有毒的化学试剂工作，产生的危险废物比一些非生物催化剂低。许多酶被用来制造单异构胺，但最近发现了一类新的酶-亚胺还原酶(IREDS)，它为合成这些重要化合物提供了新的和改进的方法。其中一个主要优势与反应产率有关；虽然许多竞争的酶路线只产生50%的最高理论产率，但IREDS可以提供所需的单一异构体形式的100%的产率。在这个项目中，我们计划开展IRED的早期工作，并对其进行开发，以期为工业提供有价值的新型酶催化剂。我们将首先改造常用的工业细菌来制造大量的已连接的酶，然后我们将彻底测试它们催化感兴趣的工业反应的能力。我们将使用X射线结晶学来确定这些酶的分子结构，这将使我们能够在分子水平上得出它们是如何工作的结论。如果我们更多地了解酶的工作原理，我们就可以使用蛋白质工程技术使酶在感兴趣的反应中更好地发挥作用，同时还可以扩大它们的适用性，使它们在不被天然酶催化的新反应中发挥作用。我们还可以利用结构信息来改进酶在工业工艺条件下的表现方式。最后，将对这类红外发光二极管进行全面审查，从而获得关于一组基本上未被探索的酶的重要新信息，以及生产重要工业合成中间体的有价值的催化剂。

The synthesis of biologically active pharmaceutical and agrochemical compounds requires that chemical intermediates are synthesised in a very specific way, giving only one of two possible isomeric forms, as the unwanted form may be inactive or even toxic. Some of the most important chemical intermediates in pharmaceuticals synthesis are amines, nitrogen containing compounds that form part of well-known drugs such as beta-blockers and painkillers such as ibuprofen. It is a significant challenge to synthesise amines in single isomer form, and increasingly both academics and industrialists are turning to enzymes - nature's biological catalysts - as an efficient way of making these compounds. Not only do enzymes naturally possess the required selectivity to make single isomers, they are also attractive from the perspective of sustainable, green chemistry, as they work at ambient temperature and pressure, do not require toxic chemical reagents to work, and generate lower hazard waste than some non-biological catalysts. Many kinds of enzyme have been used to make single-isomer amines, but a new class of enzymes 'imine reductases (IREDs)' has recently been unearthed, which offers new and improved ways to synthesise these important compounds. One of the major advantages relates to reaction yield; while many of the competing enzymatic routes result in only 50% maximal theoretical yield, IREDs can deliver 100% yield of the single isomer form required. In this project we propose to take our early work on IREDs and to develop this with a view to offering valuable new enzyme catalysts for industry. We will first engineer commonly used industrial bacteria to make large amounts of the IRED enzymes, and then we will thoroughly test their ability to catalyse the industrial reactions of interest. We will use X-ray crystallography to determine the molecular structure of these enzymes, and this will allow us to make conclusions about how they work at a molecular level. If we learn more about how the enzymes work, we can then use protein engineering techniques to make the enzymes work better on the reactions of interest, but also expand their applicability so that they work on new reactions that are not catalysed by the natural enzymes. We can also use the structural information to improve the way that the enzymes will perform under industrial process conditions. In the end, a comprehensive review of this class of IREDs will be performed, resulting in important new information on a largely unexplored group of enzymes, and valuable catalysts for the production of important industrial synthetic intermediates.