Biochem. J. (2006) 395
(191–201) (Printed in Great Britain)
Heterologous production in Wolinella succinogenes and characterization of the quinol:fumarate reductase enzymes from Helicobacter pylori and Campylobacter jejuni
Mauro MILENI*, Fraser MacMILLAN†, Christos TZIATZIOS‡
1, Klaus ZWICKER§, Alexander H. HAAS*, Werner MÄNTELE‡, Jörg SIMON
and 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, †Institute of Physical and Theoretical Chemistry, J.W. Goethe University, Marie-Curie-Str. 11, 60439 Frankfurt am Main, Germany, ‡Institute of Biophysics, J.W. Goethe University, Max-von-Laue-Str. 1, 60438 Frankfurt am Main, Germany, §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, and
Institute of Molecular Biosciences, J.W. Goethe University, Marie-Curie-Str. 9, 60439 Frankfurt am Main, Germany
The e-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 e-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.
Key words: Campylobacter jejuni, Helicobacter pylori, heterologous gene expression, membrane protein purification, quinol:fumarate reductase, Wolinella succinogenes.
Abbreviations used: BV, benzyl viologen; cat, chloramphenicol acetyltransferase; cw-EPR, continuous-wave EPR; DMN, 2,3-dimethyl-1,4-naphthoquinone; DMNH2, 2,3-dimethyl-1,4-naphthoquinol; MALT, mucosa-associated lymphoid tissue; MB, Methylene Blue; MK, menaquinone; QFR, quinol:fumarate reductase; SQOR, succinate:quinone oxidoreductase; SQR, succinate:quinone reductase.
1Present address: Technical Education Institute of Larissa, 411 10 Larissa, Greece.
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).
The frdCAB sequence from pCatCj4 reported will appear in DDBJ, EMBL, GenBank® and GSDB Nucleotide Sequence Databases under the accession number AJ628040.
Received 18 October 2005/15 December 2005; accepted 21 December 2005
Published as BJ Immediate Publication 21 December 2005, doi:10.1042/BJ20051675
The Biochemical Society, London ©2006
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