title: Site directed mutagenesis studies of FAD-dependent glucose dehydrogenase catalytic subunit of Burkholderia cepacia.
abstract: A FAD-dependent glucose dehydrogenase (FADGDH) mutant with narrow substrate specificity was constructed by site-directed mutagenesis. Several characteristics of FADGDH, such as high catalytic activity and high electron transfer ability, make this enzyme suitable for application to glucose sensors. However, for further applications, improvement of the broad substrate specificity is needed. In this paper, we mutated two residues, Asn475 and Ala472, which are located near the putative active site of the catalytic subunit of FADGDH and have been predicted from the alignment with the active site of glucose oxidase. Of the 38 mutants constructed, Ala472Phe and Asn475Asp were purified and their activities were analyzed. Both mutants showed a higher specificity toward glucose compared to the wild type enzyme.
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16, Naka-cho, Koganei, Tokyo, 184-8588, Japan.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Yamaoka
authors.forename: Yuki
authors.initials: Y
authors.lastname: Yamashita
authors.forename: Stefano
authors.initials: S
authors.lastname: Ferri
authors.forename: Koji
authors.initials: K
authors.lastname: Sode
mesh_terms: D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D001216:Asparagine / Q000235:genetics; D001483:Base Sequence; D016956:Burkholderia cepacia / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D042843:Glucose 1-Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D005182:Flavin-Adenine Dinucleotide; D001216:Asparagine; D042843:Glucose 1-Dehydrogenase; D005947:Glucose; D000409:Alanine