Mutant isolation of the Escherichia coli quinoprotein glucose dehydrogenase and analysis of crucial residues Asp-730 and His-775 for its function.
Several mutants of quinoprotein glucose dehydrogenase (GDH) in Escherichia coli were obtained and characterized. Of these, significant mutants were further characterized by kinetic analysis after purification or by site-directed mutagenesis to introduce different amino acid substitutions. H775R and H775A showed a pronounced reduction of affinity for a prosthetic group, pyrroloquinoline quinone (PQQ), suggesting that His-775 may directly interact with PQQ. D730N and D730A showed low glucose oxidase activity without influence on the affinity for PQQ, Mg2+, or substrate, but D730R showed reduced affinity for PQQ. The spectrum of tryptophan fluorescence revealed that the local structure surrounding PQQ was not changed by D730N mutation. Based on these data, we assume that Asp-730 may occur close to PQQ and function as a proton (and also electron) donor to PQQ or acceptor from PQQH2. Substitutions of Gly-689, that are located at the end of a unique segment of GDH among homologous quinoprotein dehydrogenases, directed reduction of the affinity for PQQ or GDH activity. Therefore, the unique segment and Asp-730 may play a specific role for GDH, which might be related to the intramolecular electron transfer from PQQ to ubiquinone.
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan. yamada@agr.yamaguchi-u.ac.jp
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Yamada
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Inbe
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Tanaka
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Sumi
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Matsushita
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Adachi
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D005948:Glucose Dehydrogenases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment
D001224:Aspartic Acid; D006639:Histidine; D005948:Glucose Dehydrogenases; C033955:glucose dehydrogenase (pyrroloquinoline-quinone); D008274:Magnesium