Reductive dehalogenases: structure, mechanism and application

还原脱卤酶：结构、机制和应用

• 批准号: BB/M007316/1
• 负责人: David Leys
• 金额: $ 45.17万
• 依托单位: University of Manchester
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2015
• 资助国家: 英国
• 起止时间: 2015 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FM007316%2F1
• 关键词: Reductive; dehalogenases; structure; mechanism; application

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.

许多酶使用辅因子(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，它含有一个被更大的有机分子包裹的钴离子。B12分子可以催化其他非常困难的转化，一系列不同的酶已经进化出来利用这一点。根据它们的催化作用的性质，它们可以分为三大类。虽然人们现在对前两类了解很多，但关于第三类，即所谓的还原脱卤酶的详细信息却很少。这些酶在还原过程(即需要电子)中催化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根本上说是有趣的，而且也有明显的应用。化学工业中有很大一部分使用卤化分子，有些用作中间体，大多数用作最终产品。卤素的存在赋予了最终产品有用的性质，但可悲的是，也经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，当在食物链中发现它们时，它们经常会得到重大新闻报道。某些含有还原脱卤酶的细菌被发现可以从这类污染物中去除卤素，但它们通常生长得太慢，或者需要高度特定的条件才能做到这一点。了解这些酶是如何实现这些转化的，将使我们能够评估这些酶的真实范围以及产生它们的微生物。经过几年的前期工作，我们最近成功地获得了第一张还原脱卤酶的原子分辨率图像。这揭示了它的许多基本性质，并提出了这种酶工作的可能机制。我们将利用这一发现以及它为我们提供的工具来解开这一机制。这将利用以蛋白质结晶学、生物物理学和分子生物学为中心的跨学科方法，以及计算机模型研究。这将决定这个酶家族的底物专一性和范围，换句话说：什么类型的分子可以被脱卤化？我们还将确定详细的机制，使我们能够准确地确定成功催化所必需的条件(例如，氧气的存在是否有影响)。随后，我们将进行一系列原则验证实验，旨在评估在生物传感和/或生物修复方面的应用范围(我们将使用溴化除草剂作为测试组件)。最后，将测试这些酶是否可以用于生物催化应用：还原脱卤化和更重要的是反向反应(氧化卤化)都是有意义的，因为它们很难以特定和绿色的方式催化。

项目成果

Catabolic Reductive Dehalogenase Substrate Complex Structures Underpin Rational Repurposing of Substrate Scope.

• DOI: 10.3390/microorganisms8091344
• 发表时间: 2020-09-02
• 期刊: Microorganisms
• 影响因子: 4.5
• 作者: Halliwell T;Fisher K;Payne KAP;Rigby SEJ;Leys D
• 通讯作者: Leys D

Epoxyqueuosine Reductase Structure Suggests a Mechanism for Cobalamin-dependent tRNA Modification.

环氧二糖苷还原酶结构提出了钴胺素依赖性tRNA修饰的机制。

• DOI: 10.1074/jbc.m115.685693
• 发表时间: 2015-11-13
• 期刊: The Journal of biological chemistry
• 作者: Payne KA;Fisher K;Sjuts H;Dunstan MS;Bellina B;Johannissen L;Barran P;Hay S;Rigby SE;Leys D
• 通讯作者: Leys D

Heterologous expression of cobalamin dependent class-III enzymes.

• DOI: 10.1016/j.pep.2020.105743
• 发表时间: 2021-01
• 期刊: Protein expression and purification
• 影响因子: 1.6
• 作者: Halliwell T;Fisher K;Payne KAP;Rigby SEJ;Leys D
• 通讯作者: Leys D