Reductive dehalogenases: structure, mechanism and application

点击翻译按钮获取中文摘要

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.