Difference between revisions of "Part:BBa K1465102"

Revision as of 23:24, 17 October 2014

Fumarate reductase (frd) from E. coli under the control of the T7 promoter

Fumerate reductase (frd) from E. coli under the control of the T7 promoter

Usage and Biology

Fumarate reductase is part of the anaerobic fumarate respiration in E. coli. It catalyzes the reaction from fumarate into succinate using fumarate as a final electron acceptor in anaerobic fumarate respiration. The related enzyme in aerobic respiration is the succinate dehydrogenase, which catalyses the reaction from succinate to fumarate. The electrons are transferred from succinate to FAD⁺ producing fumarate and FADH₂. The succinate dehydrogenase is also a membrane enzyme and it is part of the citric acid cycle. They both belong to the respiration complex II. Naturally there is no activity of both enzymes at the same time in E. coli cells. (Iverson et al., 1999)
In our project we used the fumarate reductase in combination with an extracellular mediator as electron donor to transfer electrons into bacterial cells. The reduced mediator crosses the outer membrane of E. coli through the outer membrane porine OprF (BBa_K1172507). Mediators adsorb at the inner membrane and transfer electrons to the fumarate reductase. After that the reduced fumarate reductase transfer electrons to fumarate producing succinate. This process has been shown for the fumarate reductase in Actinobacillus succinogenes by Park et al..
Succinate can serve as a substrate for the succinate dehydrogenase, which catalyzes the oxidation of succinate into fumarate again. So we want to create a loop in the citric acid cycle between fumarate and succinate generating FADH₂ as a reductive power in the cell. Electrons are transferred to FAD+, which generate proton translocation from the cytoplasm into the periplasmatic space. The proton motoric force leads to ATP production. To conclude the mediator-dependent activity of the fumarate reductase could serve as an energy source for bacterial cells.
We plan on comparing the activity of the fumarate reductases (Frd) from Escherichia coli KRX and the fumarate reductase (Fum)from Actinobacillus succinogenes working with different mediators, for example neutral red or bromphenol blue, as electron donors.

Sequence and Features