C-terminal periplasmic domain of Escherichia coli quinoprotein glucose dehydrogenase transfers electrons to ubiquinone.
Membrane-bound quinoprotein glucose dehydrogenase (GDH) in Escherichia coli donates electrons directly to ubiquinone during the oxidation of d-glucose as a substrate, and these electrons are subsequently transferred to ubiquinol oxidase in the respiratory chain. To determine whether the specific ubiquinone-reacting site of GDH resides in the N-terminal transmembrane domain or in the large C-terminal periplasmic catalytic domain (cGDH), we constructed a fusion protein between the signal sequence of beta-lactamase and cGDH. This truncated GDH was found to complement a GDH gene-disrupted strain in vivo. The signal sequence of the fused protein was shown to be cleaved off, and the remaining cGDH was shown to be recovered in the membrane fraction, suggesting that cGDH has a membrane-interacting site that is responsible for binding to membrane, like peripheral proteins. Kinetic analysis and reconstitution experiments revealed that cGDH has ubiquinone reductase activity nearly equivalent to that of the wild-type GDH. Thus, it is likely that the C-terminal periplasmic domain of GDH possesses a ubiquinone-reacting site and transfers electrons directly to ubiquinone.
Elias
M
M
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan.
Tanaka
M
M
Sakai
M
M
Toyama
H
H
Matsushita
K
K
Adachi
O
O
Yamada
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D004579:Electron Transport; D004926:Escherichia coli / Q000201:enzymology*; D019143:Evolution, Molecular; D005816:Genetic Complementation Test; D005948:Glucose Dehydrogenases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005949:Glucose Oxidase / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D019897:Periplasm / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014451:Ubiquinone / Q000378:metabolism*; D001618:beta-Lactamases / Q000378:metabolism
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D014451:Ubiquinone; D005948:Glucose Dehydrogenases; D005949:Glucose Oxidase; C033955:glucose dehydrogenase (pyrroloquinoline-quinone); D001618:beta-Lactamases