title: Engineering pyranose 2-oxidase for modified oxygen reactivity.
abstract: Pyranose 2-oxidase (POx), a member of the GMC family of flavoproteins, catalyzes the regioselective oxidation of aldopyranoses at position C2 to the corresponding 2-ketoaldoses. During the first half-reaction, FAD is reduced to FADH2 and reoxidized in the second half-reaction by reducing molecular oxygen to H2O2. Alternative electron acceptors including quinones, radicals or chelated metal ions show significant and in some cases even higher activity. While oxygen as cheap and abundantly available electron acceptor is favored for many processes, reduced oxygen reactivity is desirable for some applications such as in biosensors/biofuel cells because of reduced oxidative damages to the biocatalyst from concomitant H2O2 production as well as reduced electron "leakage" to oxygen. The reactivity of flavoproteins with oxygen is of considerable scientific interest, and the determinants of oxygen activation and reactivity are the subject of numerous studies. We applied site-saturation mutagenesis on a set of eleven amino acids around the active site based on the crystal structure of the enzyme. Using microtiter plate screening assays with peroxidase/2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) and 2,6-dichlorophenolindophenol, variants of POx with decreased oxidase activity and maintained dehydrogenase activity were identified. Variants T166R, Q448H, L545C, L547R and N593C were characterized with respect to their apparent steady-state constants with oxygen and the alternative electron acceptors DCPIP, 1,4-benzoquinone and ferricenium ion, and the effect of the mutations was rationalized based on structural properties.
authors.affiliation: Food Biotechnology Laboratory, Department of Food Sciences and Technology, BOKU - University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Dagmar
authors.initials: D
authors.lastname: Brugger
authors.affiliation: Food Biotechnology Laboratory, Department of Food Sciences and Technology, BOKU - University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Iris
authors.initials: I
authors.lastname: Krondorfer
authors.affiliation: Food Biotechnology Laboratory, Department of Food Sciences and Technology, BOKU - University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Christopher
authors.initials: C
authors.lastname: Shelswell
authors.affiliation: Food Biotechnology Laboratory, Department of Food Sciences and Technology, BOKU - University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Huber-Dittes
authors.affiliation: Food Biotechnology Laboratory, Department of Food Sciences and Technology, BOKU - University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Dietmar
authors.initials: D
authors.lastname: Haltrich
authors.affiliation: Food Biotechnology Laboratory, Department of Food Sciences and Technology, BOKU - University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Clemens K
authors.initials: CK
authors.lastname: Peterbauer
mesh_terms: D016227:Benzoquinones / Q000378:metabolism; D002237:Carbohydrate Dehydrogenases / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D013379:Substrate Specificity
chemical_list: D016227:Benzoquinones; C004532:quinone; D006861:Hydrogen Peroxide; D002237:Carbohydrate Dehydrogenases; C022489:pyranose oxidase; D010100:Oxygen