Jan Kägi

Abstract

The aerobic respiratory chain of Escherichia coli is the most important energy transportation pathway and is used for the production of ATP. The cytochrome bd-II oxidase is an integral membrane protein of E. coli and many other prokaryotes. It is one of the terminal oxidases of the aerobic respiratory chain and is expressed under very low oxygen tension and under anaerobic conditions. This allows E. coli to adapt quickly to changing environmental conditions. The enzyme transfers two electrons from a quinone pool to oxygen, which is reduced to water. This is coupled with the generation of a proton gradient across the inner cell membrane, which is used for the production of ATP. Since many pathogenic prokaryotes possess the bd-II oxidase, which plays a central role in their organism, it is an interesting target for drug development. The three-dimensional structure of the E. coli cytochrome bd-II oxidase, which is essential for the understanding of the enzyme, has not been determined so far. The main aim of this project is to characterize and understand the function of the cytochrome bd-II oxidase of E. coli. For this purpose, the cytochrome bd-II oxidase genes of will be cloned into a vector for overexpression. In the further course the cytochrome bd-II oxidase will be homologously overproduced and purified. The three-dimensional structure is to be determined by protein crystallization or cryo-electron microscopy.

Methods

• Manipulation of genes (AQUA cloning, RF cloning)
• Heterologous production
• Purification and characterization of proteins
• Reconstitution of proteins in liposomes
• Fluorescence and UV/VIS spectroscopy
• Enzyme kinetics

Publications

Theßeling A, Rasmussen T, Burschel S, Wohlwend D, Kägi J, Müller R, Böttcher B and Friedrich T (2019) Homologous bd oxidases share the same architecture but differ in mechanism. Nature Communications 10, 5138. doi:10.1038/s41467-019-13122-4