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Towards unravelling the mechanism of catabolic reductive dehalogenases

Halliwell, Thomas David

[Thesis]. Manchester, UK: The University of Manchester; 2020.

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Abstract

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.

Bibliographic metadata

Type of resource:
Content type:
Administered thesis
Form of thesis:
Traditional
Type of submission:
Doctoral level ETD - final
Thesis title:
Towards unravelling the mechanism of catabolic reductive dehalogenases
Degree type:
Doctor of Philosophy
Degree programme:
BBSRC Doctoral Training Programme Chemistry
Publication date:
2020-02-10T11:41:58
Institution:
The University of Manchester
Location:
Manchester, UK
Total pages:
187
Abstract:
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.
Keyword(s):
Thesis main supervisor(s):
Thesis co-supervisor(s):
Degree grantor:
Language:
en

Institutional metadata

University researcher(s):
Academic department(s):

Record metadata

Manchester eScholar ID:
uk-ac-man-scw:323612
Created by:
Halliwell, Thomas
Created:
10th February, 2020, 11:41:58
Last modified by:
Halliwell, Thomas
Last modified:
2nd March, 2021, 10:58:19

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