title: Construction of mutant glucose oxidases with increased  dye-mediated dehydrogenase activity.
abstract: Mutagenesis studies on glucose oxidases (GOxs) were conducted to  construct GOxs with reduced oxidase activity and increased dehydrogenase activity.  We focused on two representative GOxs, of which crystal structures have already been  reported&#8212;Penicillium amagasakiense GOx (PDB ID; 1gpe) and Aspergillus niger GOx (PDB ID; 1cf3). We constructed oxygen-interacting structural models for GOxs, and predicted the residues responsible for oxidative half reaction with oxygen on the basis of the crystal structure of cholesterol oxidase as well as on the fact that both enzymes are members of the glucose/methanol/choline (GMC) oxidoreductase family. Rational amino acid substitution resulted in the construction of an engineered GOx with drastically decreased oxidase activity and increased dehydrogenase activity, which was higher  than that of the wild-type enzyme. As a result, the dehydrogenase/oxidase ratio of the engineered enzyme was more than 11-fold greater than that of the wild-type enzyme. These results indicate that alteration of the dehydrogenase/oxidase activity ratio of GOxs is possible by introducing a mutation into the putative functional residues responsible for oxidative half reaction with oxygen of these enzymes, resulting in a further increased dehydrogenase activity. This is the first study reporting the alteration of GOx electron acceptor preference from oxygen to an artificial electron acceptor.
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei, Tokyo 184-8588, Japan. sode@cc.tuat.ac.jp.
authors.forename: Yohei
authors.initials: Y
authors.lastname: Horaguchi
authors.forename: Shoko
authors.initials: S
authors.lastname: Saito
authors.forename: Katsuhiro
authors.initials: K
authors.lastname: Kojima
authors.forename: Wakako
authors.initials: W
authors.lastname: Tsugawa
authors.forename: Stefano
authors.initials: S
authors.lastname: Ferri
authors.forename: Koji
authors.initials: K
authors.lastname: Sode
mesh_terms: D019943:Amino Acid Substitution; D004396:Coloring Agents / Q000737:chemistry / Q000378:metabolism*; D005949:Glucose Oxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008968:Molecular Conformation; D009154:Mutation; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D004396:Coloring Agents; D010088:Oxidoreductases; D005949:Glucose Oxidase; D010100:Oxygen