Heterologous production in Wolinella succinogenes and characterization of the quinol:fumarate reductase enzymes from Helicobacter pylori and Campylobacter jejuni.
The epsilon-proteobacteria Helicobacter pylori and Campylobacter jejuni are both human pathogens. They colonize mucosal surfaces causing severe diseases. The membrane protein complex QFR (quinol:fumarate reductase) from H. pylori has previously been established as a potential drug target, and the same is likely for the QFR from C. jejuni. In the present paper, we describe the cloning of the QFR operons from the two pathogenic bacteria H. pylori and C. jejuni and their expression in Wolinella succinogenes, a non-pathogenic -proteobacterium. To our knowledge, this is the first documentation of heterologous membrane protein production in W. succinogenes. We demonstrate that the replacement of the homologous enzyme from W. succinogenes with the heterologous enzymes yields mutants where fumarate respiration is fully functional. We have isolated and characterized the heterologous QFR enzymes. The high quality of the enzyme preparation enabled us to determine unequivocally by analytical ultracentrifugation the homodimeric state of the three detergent-solubilized heterotrimeric QFR enzymes, to accurately determine the different oxidation-reduction ('redox') midpoint potentials of the six prosthetic groups, the Michaelis constants for the quinol substrate, maximal enzymatic activities and the characterization of three different anti-helminths previously suggested to be inhibitors of the QFR enzymes from H. pylori and C. jejuni. This characterization allows, for the first time, a detailed comparison of the QFR enzymes from C. jejuni and H. pylori with that of W. succinogenes.
Mileni
Mauro
M
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max-von-Laue-Str. 3, 60438 Frankfurt am Main, Germany.
MacMillan
Fraser
F
Tziatzios
Christos
C
Zwicker
Klaus
K
Haas
Alexander H
AH
Mäntele
Werner
W
Simon
Jörg
J
Lancaster
C Roy D
CR
D016123:Campylobacter jejuni / Q000201:enzymology* / Q000235:genetics; D003067:Coenzymes / Q000378:metabolism; D016480:Helicobacter pylori / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009876:Operon / Q000235:genetics; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D013057:Spectrum Analysis; D014461:Ultracentrifugation; D016968:Wolinella / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*
D003067:Coenzymes; D010088:Oxidoreductases; C424907:quinol fumarate reductase