Reductive dehalogenases: structure, mechanism and application

许多酶使用辅助因子（通常是维生素）来实现它们催化的分子转化。其中之一是B12，这是一种相当复杂的分子，它包含一个钴离子，被一个更大的有机分子包裹着。B12分子可以催化其他非常困难的转化，并且已经进化出一系列不同的酶来利用它。根据它们的催化性质，它们可以分为三大类。虽然现在对前两组已经了解很多，但关于第三组，即所谓的还原脱卤酶的详细信息却很少。这些酶催化在还原过程中（即需要电子）从有机分子中去除卤素原子（氯、溴或碘）。这种特殊的反应不仅从基本观点来看很有趣，而且有明显的应用。化学工业的很大一部分使用卤化分子，有些作为中间体，大多数作为最终产品。卤素的存在给最终产品带来了有用的特性，但令人遗憾的是，它也常常导致对环境的有毒影响。一些更臭名昭著的污染物是多氯联苯或二恶英，当它们在食物链中被发现时，经常会得到大量的新闻报道。人们发现含有还原脱卤酶的某些细菌可以从这些污染物中去除卤素，但它们通常生长得太慢，或者需要非常特殊的条件才能做到这一点。了解酶如何实现这些转化将使我们能够评估这些酶和产生它们的微生物的真正范围。经过几年的初步工作，我们最近成功地获得了还原脱卤酶的第一张原子分辨率图片。这揭示了它的许多基本特性，并提出了这种酶工作的可能机制。我们将利用这一发现及其提供的工具来解开这一机制。这将利用以蛋白质晶体学、生物物理学和分子生物学为中心的跨学科方法，以及计算机建模研究。这将决定底物的特异性和该酶家族的范围，换句话说：什么类型的分子可以去卤？我们还将确定详细的机制，使我们能够确定成功催化所必需的条件（例如，氧气的存在是否有影响）。在此之后，我们将进行一系列的原理验证实验，旨在评估生物传感和/或生物修复的应用范围（我们将使用溴化除草剂作为测试成分）。最后，我们将测试这些酶是否可以用于生物催化应用：还原性脱卤化和更重要的逆反应（氧化卤化）都是我们感兴趣的，因为这些反应很难以特定的绿色方式催化。

Many enzymes use cofactors (often these are vitamins) to achieve the molecular transformations they catalyse. One of these is B12, a rather complex molecule that contains a cobalt ion encapsulated by a larger organic molecule. The B12 molecule can catalyse otherwise very difficult transformations, and a range of different enzymes have evolved to make use of this. These can be classed in three broad groups, dependent on the nature of their catalysis. While much is now known about the first 2 groups, there has been little detailed information about the third, the so-called reductive dehalogenases. These enzymes catalyse the removal of a halogen atom (chloride, bromide or iodide) from an organic molecule in a reductive process (ie requiring electrons). This particular reaction is not only interesting from a fundamental point of view, but also has obvious applications. A large proportion of the chemical industry makes use of halogenated molecules, some as intermediates, most as end product. The presence of the halogen confers useful properties to the end product, but sadly often also leads to toxic effects for the environment. Some of the more infamous pollutants are PCBs or dioxins, which regularly get significant news coverage when discovered in the food chain. Certain bacteria that contains reductive dehalogenases have been found to remove the halogens from such pollutants, but they often grow too slowly or require highly specific conditions to do so. Understanding how the enzymes achieve these transformation would allow us to assess the true scope of these enzymes and the microorganisms that produce them. Following several years of preliminary work, we have recently managed to get the first atomic resolution picture of a reductive dehalogenase. This is revealed many of its fundamental properties, and suggested possible mechanisms by which this enzyme works. We will capitalise on this discovery and the tools it offers us to unravel that mechanism. This will make use of an interdisciplinary approach centred around protein crystallography, biophysics and molecular biology alongside computer modelling studies. This will determine substrate specificity and scope for this enzyme family, in other words: what type of molecules can be dehalogenated? We will also determine the detailed mechanism, allowing us to pinpoint what conditions will be necessary for successful catalysis (whether presence of oxygen has an effect for example). Following this, we will conduct a series of proof-of-principle experiments that are aimed at assessing the scope for application in biosensing and/or bioremediation (we will use a brominated herbicide as test component). Finally, will test whether these enzyme can be used in biocatalysis applications: both the reductive dehalogenation and more importantly the reverse reaction (oxidative halogenation) are of interest as these are difficult to catalyse in a specific and green manner.