Quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa: the unusual disulfide ring formed by adjacent cysteine residues is essential for efficient electron transfer to cytochrome c 550
All pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenases contain an unusual disulfide 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 Heterologously expressed quinoprotein ethanol dehydrogenase in which Cys-105 and Cys-106 have been replaced by Ala (Cys105Ala/Cys106Ala apo-QEDH) was successfully converted to enzymatic active holo-enzyme by incorporation of its cofactor PQQ in the presence of Ca2+.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 artificial 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 affinity for the secondary alcohol 2-propanol was unaltered. Surprisingly, for all substrates tested the specific activity of the mutant enzyme in the artificial dye test was higher than that found for wild type QEDH. On the contrary, in the ferricyanide test with the natural electron acceptor cytochrome c 550 the activity of mutant Cys105Ala/Cys106Ala was 15-fold lower than that of wild type QEDH. We demonstrate for the first time unambiguously that the unusual disulfide ring is essential for efficient electron transfer at pH 7 from QEDH to its natural electron acceptor cytochrome c 550.
KeywordsActive site Adjacent Cys/Ala replacement Pyrroloquinoline quinone (PQQ) QEDH Site directed mutagenesis
Quinoprotein ethanol dehydrogenase
Quinoprotein ethanol dehydrogenase in which Cys-105 and Cys-106 have been replaced by Ala
Quinoprotein methanol dehydrogenase
N-Methylphenazonium methyl sulfate
Luria broth growth medium
Electron paramagnetic resonance
We thank Mrs. I. Maue-Mohn for expert technical assistance. This work was supported in part by grant Go 15-2 from the Deutsche Forschungsgemeinschaft, Bonn, Germany. Plasmid pEC86 was kindly provided by Mrs. Thöny-Meyer, Eidgenössische Technische Hochschule Zürich.
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