Imine Reductases: Biochemistry, Engineering and Application

生物活性药物和农业化学化合物的合成需要以非常特定的方式合成化学中间体，仅得到两种可能的异构体形式之一，因为不需要的形式可能是无活性的或甚至有毒的。在药物合成中，一些最重要的化学中间体是胺，含氮化合物，其形成众所周知的药物的一部分，如β-受体阻滞剂和止痛药，如布洛芬。以单一异构体形式合成胺是一个重大挑战，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有效方法。酶不仅天然地具有制备单一异构体所需的选择性，而且从可持续的绿色化学的角度来看，它们也是有吸引力的，因为它们在环境温度和压力下工作，不需要有毒的化学试剂来工作，并且产生比一些非生物催化剂更低的危险废物。许多种酶已被用于合成单异构体胺，但最近发现了一类新的酶“亚胺还原酶（IREDs）”，这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;虽然许多竞争性酶促途径的最大理论产率仅为50%，但IRED可以提供所需单一异构体形式的100%产率。在这个项目中，我们建议采取我们的早期工作的IRED和发展，以期提供有价值的新的酶催化剂的工业。我们将首先设计常用的工业细菌来制造大量的IRED酶，然后我们将彻底测试它们催化工业反应的能力。我们将使用X射线晶体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更好地作用于感兴趣的反应，而且还可以扩展它们的适用性，使它们作用于天然酶催化不到的新反应。我们还可以使用结构信息来改善酶在工业过程条件下的表现方式。最后，这类IRED将进行全面审查，导致在很大程度上未开发的一组酶的重要的新信息，和有价值的催化剂，用于生产重要的工业合成中间体。

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.