Abstract
Topological structure of quinoprotein glucose dehydrogenase in the inner membrane of Escherichia coli was determined by constructing protein fusions with alkaline phosphatase or beta-galactosidase. Analysis of the fusions revealed that the dehydrogenase possesses five membrane-spanning segments, and the N-terminal and C-terminal portions resided at the cytoplasmic and periplasmic side of the membrane, respectively. These results agreed with the hydropathy profile based on its primary structure. The topological structure suggests that the predicted binding site of the prosthetic group pyrroloquinoline quinone is located at the periplasmic side and that the amino acid residues corresponding to those that were presumed to interact with ubiquinone in one subunit of mitochondrial NADH dehydrogenase also occur at the periplasmic side. When the purified glucose dehydrogenase and cytochrome o ubiquinol oxidase were reconstituted together with ubiquinone into liposomes, a membrane potential could be generated by the electron transfer at the site of the ubiquinol oxidase but not of the dehydrogenase. These results suggest that glucose dehydrogenase has a ubiquinone reacting site close to the periplasmic side of the membrane, and thus its electron transfer to ubiquinone appears to be incapable of forming a proton electrochemical gradient across the inner membrane of E. coli.
Highlights
Topological structure of quinoprotein glucose dehy- gene encoding glucose dehydrogenase in E. coli is finely regdrogenase in the inner membrane of Escherichia coli ulated [6]. was determined by constructing protein fusionswith Molecular genetic and biochemical analyses of several dealkaline phosphatase or @-galactosidase
To cytochrome oxidase through ubiquinone in therespiratory In thispaper, we have determined the topological structure chain [1].The membrane-bound glucose dehydrogenase is of the E. coli glucose dehydrogenase to establish the basis for known to be a quinoprotein requiring a novel cofactor, PQQ,’ clarifying its functionaldomains
T o define the topological structure of the with alkaline phosphatase connected to the middle portion proteininthemembrane, we fundamentally utilized the and the C-terminal portion of method established by Manoil and Beckwith[15] and devel- glucose dehydrogenase
Summary
In thiscase, @galactosidase activities from cells harboring odd fusion plasmids were significantly higher than those frcoemlls harboring even (Fig. 3). T o define the topological structure of the with alkaline phosphatase connected to the middle portion proteininthemembrane, we fundamentally utilized the (at the EcoRV) and the C-terminal portion (at the BglI) of method established by Manoil and Beckwith[15] and devel- glucose dehydrogenase. Both fusions exhibited blue colonies opeda simple and correct fusion procedure by using PCR and high enzyme activities (data not shown).
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