Quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa : the unusual disulfide ring formed by adjacent cysteine
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ORIGINAL PAPER
Quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa: the unusual disulWde ring formed by adjacent cysteine residues is essential for eYcient electron transfer to cytochrome c550 Bina Mennenga · Christopher W. M. Kay · Helmut Görisch
Received: 29 October 2008 / Revised: 21 January 2009 / Accepted: 26 January 2009 / Published online: 18 February 2009 © Springer-Verlag 2009
Abstract All pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenases contain an unusual disulWde ring formed between adjacent cysteine residues. A mutant enzyme that is lacking this structure was generated by replacing Cys105 and Cys106 with Ala in quinoprotein ethanol dehydrogenase (QEDH) from Pseudomonas aeruginosa ATCC17933. Heterologously expressed quinoprotein ethanol dehydrogenase in which Cys-105 and Cys-106 have been replaced by Ala (Cys105Ala/Cys106Ala apoQEDH) was successfully converted to enzymatic active holo-enzyme by incorporation of its cofactor PQQ in the presence of Ca2+. The enzymatic activity of the mutant enzyme in the artiWcial dye test with N-methylphenazonium methyl sulfate (PMS) and 2,6-dichlorophenol indophenol (DCPIP) at pH 9 did not depend on an activating amine which is essential for wild type activity under these conditions. The mutant enzyme showed increased Michaelis constants for primary alcohols, while the aYnity for the secondary alcohol 2-propanol was unaltered. Surprisingly, for all substrates tested the speciWc activity of the mutant enzyme in the artiWcial dye test was higher than that found for wild type QEDH. On the contrary, in the ferricyanide
test with the natural electron acceptor cytochrome c550 the activity of mutant Cys105Ala/Cys106Ala was 15-fold lower than that of wild type QEDH. We demonstrate for the Wrst time unambiguously that the unusual disulWde ring is essential for eYcient electron transfer at pH 7 from QEDH to its natural electron acceptor cytochrome c550. Keywords Active site · Adjacent Cys/Ala replacement · Pyrroloquinoline quinone (PQQ) · QEDH · Site directed mutagenesis Abbreviations PQQ Pyrroloquinoline quinone QEDH Quinoprotein ethanol dehydrogenase Cys105Ala/ Quinoprotein ethanol dehydrogenase in Cys106Ala which Cys-105 and Cys-106 have been replaced by Ala QMDH Quinoprotein methanol dehydrogenase PMS N-Methylphenazonium methyl sulfate DCPIP 2,6-Dichlorophenol indophenol LB Luria broth growth medium IPTG Isopropyl--D-thiogalactopyranoside EPR Electron paramagnetic resonance
Communicated by Arnold Driessen. B. Mennenga · H. Görisch (&) Fachgebiet Angewandte Biochemie, Institut für Biotechnologie, Technische Universität Berlin, Seestrasse 13, 13353 Berlin, Germany e-mail: [email protected] C. W. M. Kay Institute of Structural and Molecular Biology, London Centre of Nanotechnology, University College London, Gower Street, London WC1E 6BT, UK
Introduction The gram-negative bacterium Pseudomonas aeruginosa ATCC17933 is able to grow on ethanol as sole source of carbon and energy. The initial step converting ethanol to acetaldehyde is catalyz
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