Reductive dehalogenases: structure, mechanism and 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.

The B12-dependent enzymes can be classed in three broad groups, depending on the nature of the chemical transformation catalysed. Group I enzymes use the homolytic cleavage of the Co-C bond in deoxyadenosyl-B12 to achieve radical catalysis, while group II enzymes use methylcobalamin to transfer methyl groups (through heterolytic cleavage of the Co-C bond). Many structures have now been determined for both groups, and detailed insights exists in the respective mechanisms. In contrast, no structure or experimentally verified mechanism exists for the group III enzymes, the reductive dehalogenases. These enzymes use a corrinoid cofactor and two 4Fe4S clusters to reductively dehalogenate a wide range of organohalide molecules. We have managed to get a heterologous source of active enzyme (a first to our knowledge), leading to our recent structure determination of the first dehalogenase. We can now capitalise on the insights this structure offers us, and the tools generated by the heterologous expression. Ultimately, we seek to understand the structure and mechanism of this enzyme and the wider enzyme family to guide and assess application. The reductive dehalogenation has obvious implications in biosensing/bioremediation of many xenobiotics, while the reaction itself as well as the reverse reaction (oxidative halogenation) could be of interest in biocatalytic approaches. We will first seek to fully characterise the enzyme for which we obtained the first crystal structure, using crystallography (ligand enzyme complexes, X-ray mediated reduction to follow reaction in crystallo), biophysics (stopped-flow, EPR, PFV) combined with computational studies (DFT calculations of active site architecture, ligand docking studies, full QM/MM simulations). Furthermore, we will attempt to broader our insights by studying a range of homologues with distinct substrate specificity. We will also use site-directed mutagenesis to probe the role of the various active site amino acids.

Beneficiaries:
The outcomes of this grant will impact on 4 main beneficiaries:
(i) synthetic biologist/enzymologists working in industry/academia- accurate and full description of reductive dehalogenase process will add to our understanding of B12-dependent enzyme function in the wider sense and add to our catalogue of available tools for further application.
(ii) The chemical industry and industrial/agricultural users - given the central role halogen chemistry plays in many industrial processes, novel abilities to specifically remove halogens at ambient conditions will be highly attractive. More importantly, the reverse reaction, specific oxidative halogenation is of considerable interest as it cannot easily be achieved. Our research and the tools it provides could give this industry the scope to develop such applications.
(iii) food industry/environmental agencies - many of the halogenated xenobiotics negatively impact on the environment, and not all release is unintentional, with many pesticides incorporating halogen atoms. This can lead to some accumulating in the food chain, leading to wide publicised contamination common food stuff by PCBs or dioxins. The use of this enzyme, either in vitro or in vivo has obvious beneficial implications as it removes the halogen from these molecules, often drastically reducing toxicity and leading to further breakdown. Such applications have already been tested, but are limited to application of the original hosts, organohalide respiring bacteria, which are strict anaerobes that can be difficult to culture. The insights we generate, as well as the enzyme variants and heterologous host(s) we will provide could significantly improve the scope of such applications.
(iv) society in the wider sense - the generation and use of organohalides at a global scale has negative impacts that are a key concern to society. This application seeks to address whether reductive dehalogenases can be used to mitigate some of these effects.

Exploitation:
We do not anticipate an immediate commercial impact, given our research is still at the fundamental stage. However, should we be able to demonstrate catalysis of the reverse reaction (oxidative halogenation) we will seek to protect the IP involved at the earliest stage. Our strategy for translating the technology is to establish IP protection through UMIP (Manchester's IP office). We will communicate through networking events with external stakeholders (industry, other University groups, venture capital groups, policy groups).

Outreach:
We anticipate wide interest in the progress and outcomes of this application given the relevance to chemical industry, and both the environment and food quality. We will make use of the internet as the tool with the widest dissemination possibilities, with a range of www based communication tools: websites, podcase and blogs regularly updated and tailored to the various types of audience being targeted (scientific, industrial or general media). In addition, we will ensure direct channels of communication with the applicants and the research staff employed through representation at UK science fairs (Big Bang, Royal Society), engage with local schools and offer summer internships in the applicants' laboratories.