Organohalides of anthropogenic origin have been produced in large quantities since the industrial revolution. However useful, these compounds are often recalcitrant to degradation and improper disposal has lead to accumulation at many contaminated sites, posing an environmental threat. The discovery of organohalide respiring bacteria, which are able to utilise organohalides for energy and growth, led to potential new applications in bioremediation. The reductive dehalogenases are crucial for the process of organohalide respiration, and these cobalamin and iron-sulphur containing enzymes are ultimately responsible for the reductive cleavage of the carbon-halogen bond. Recent insights into the structure and mechanism of these enzymes have been obtained, made possible by the (heterologous) expression of these enzymes. However, many questions regarding mechanism, substrate specificity and enzyme reduction remain. In this study we compare a range of methods for heterologous expression of the class-III cobalamin-dependent enzymes, consisting of both reductive dehalogenases and the related epoxyqueuosine reductases. We also report improved crystallisation of the soluble catabolic reductive dehalogenase NpRdhA, leading to the first substrate-bound and variant crystal structures, providing further support for the proposed mechanism of action. Finally, taking advantage of the optimised heterologous expression methods, we have been able to express and characterise a natural self-sufficient catabolic reductive dehalogenase. We reveal the latter is able to reductively debrominate a wide range of compounds both in vivo and in vitro, including flame-retardants. Our results provide a platform for the future expression and characterisation of additional reductive dehalogenases and hopefully inform future bioremediation application of these enzymes.
|Date of Award||1 Aug 2020|
- The University of Manchester
|Supervisor||David Leys (Supervisor), Sam Hay (Supervisor) & Neil Dixon (Supervisor)|
- Protein expression
- Vitamin B12