A Biochemical Study Of A Catabolic Reductive Dehalogenase

UoM administered thesis: Phd

  • Authors:
  • Fraser Collins

Abstract

Among the many complex and growing threats to the environment, the danger posed by persistent organohalide pollutants stands out. In the 20th Century, chemical manufacturers began to synthesize organohalides for use in industry, which has unfortunately led to the contamination of soils and groundwater aquifers around the world, as well as the atmosphere itself. Unfortunately, many of these organohalides are recalcitrant to degradation and highly toxic to both humans and wildlife. Persistent organohalide pollution is now a globally recognized issue, with scientific research aiming to solve the problems developed over the past century. The discovery of the existence of organohalide-respiring bacteria, which utilize organohalides for energy and growth, might lead to solutions. The potential to apply these organisms in projects that seek to remediate areas contaminated by organohalides was recognized and this led to an increased interest in the process of organohalide respiration. Central to this metabolic process are the reductive dehalogenases, an unusual group of cobalamin (vitamin-B12)- and iron-sulfur cluster-containing enzymes. The first insight into the structure and catalytic mechanism of these proteins has only recently been reported. In order for the soluble catabolic reductive dehalogenase NpRdhA to be used as a model system, a reducing system is required. We tested the proposed physiological redox partners but were unable to obtain efficient electron transfer between these and NpRdhA. We developed an alternative and efficient in vitro reducing system using non-physiological redox partners that underpins detailed solution studies. A series of NpRdhA variants were designed and characterized to verify whether the enzyme substrate specificity can be altered, without affecting catalytic and/or cofactor binding properties. Finally, a novel protein production platform for RdhAs in E. coli based on the vitamin-B12 transporter, BtuB has been developed. Our findings provide further insight into catabolic RdhA, and by extension the respiratory enzymes, and have laid the groundwork for future protein characterization and protein engineering. Such work goes to increase the potential of reductive dehalogenases to be used in future bioremediation projects.

Details

Original languageEnglish
Awarding Institution
Supervisors/Advisors
Award date1 Aug 2018