The ϵ-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 ϵ-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.
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Research Article|
March 15 2006
Heterologous production in Wolinella succinogenes and characterization of the quinol:fumarate reductase enzymes from Helicobacter pylori and Campylobacter jejuni
Mauro Mileni;
Mauro Mileni
*Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max-von-Laue-Str. 3, 60438 Frankfurt am Main, Germany
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Fraser MacMillan;
Fraser MacMillan
†Institute of Physical and Theoretical Chemistry, J.W. Goethe University, Marie-Curie-Str. 11, 60439 Frankfurt am Main, Germany
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Christos Tziatzios;
Christos Tziatzios
1
‡Institute of Biophysics, J.W. Goethe University, Max-von-Laue-Str. 1, 60438 Frankfurt am Main, Germany
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Klaus Zwicker;
Klaus Zwicker
§Department of Medicine, Institute of Molecular Bioenergetics, Gustav Embden Centre of Biological Chemistry, J.W. Goethe University, Theodor-Stern-Kai 7, Haus 25B, 60590 Frankfurt am Main, Germany
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Alexander H. Haas;
Alexander H. Haas
*Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max-von-Laue-Str. 3, 60438 Frankfurt am Main, Germany
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Werner Mäntele;
Werner Mäntele
‡Institute of Biophysics, J.W. Goethe University, Max-von-Laue-Str. 1, 60438 Frankfurt am Main, Germany
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Jörg Simon;
Jörg Simon
∥Institute of Molecular Biosciences, J.W. Goethe University, Marie-Curie-Str. 9, 60439 Frankfurt am Main, Germany
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C. Roy D. Lancaster
C. Roy D. Lancaster
2
*Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max-von-Laue-Str. 3, 60438 Frankfurt am Main, Germany
2To whom correspondence should be addressed, at Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, P.O. Box 55 03 53, 60402 Frankfurt am Main, Germany (email Roy.Lancaster@mpibp-frankfurt.mpg.de).
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Publisher: Portland Press Ltd
Received:
October 18 2005
Revision Received:
December 15 2005
Accepted:
December 21 2005
Accepted Manuscript online:
December 21 2005
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2006
Biochem J (2006) 395 (1): 191–201.
Article history
Received:
October 18 2005
Revision Received:
December 15 2005
Accepted:
December 21 2005
Accepted Manuscript online:
December 21 2005
Citation
Mauro Mileni, Fraser MacMillan, Christos Tziatzios, Klaus Zwicker, Alexander H. Haas, Werner Mäntele, Jörg Simon, C. Roy D. Lancaster; Heterologous production in Wolinella succinogenes and characterization of the quinol:fumarate reductase enzymes from Helicobacter pylori and Campylobacter jejuni. Biochem J 1 April 2006; 395 (1): 191–201. doi: https://doi.org/10.1042/BJ20051675
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