Engineering pyranose 2-oxidase for modified oxygen reactivity.

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 o...

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Autores principales:	Dagmar Brugger, Iris Krondorfer, Christopher Shelswell, Benjamin Huber-Dittes, Dietmar Haltrich, Clemens K Peterbauer
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2014
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/5fffd99c82aa4bbcad797df5e930b929
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spelling	oai:doaj.org-article:5fffd99c82aa4bbcad797df5e930b9292021-11-25T05:57:27ZEngineering pyranose 2-oxidase for modified oxygen reactivity.1932-620310.1371/journal.pone.0109242https://doaj.org/article/5fffd99c82aa4bbcad797df5e930b9292014-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0109242https://doaj.org/toc/1932-6203Pyranose 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.Dagmar BruggerIris KrondorferChristopher ShelswellBenjamin Huber-DittesDietmar HaltrichClemens K PeterbauerPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 10, p e109242 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Dagmar Brugger Iris Krondorfer Christopher Shelswell Benjamin Huber-Dittes Dietmar Haltrich Clemens K Peterbauer Engineering pyranose 2-oxidase for modified oxygen reactivity.
description	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.
format	article
author	Dagmar Brugger Iris Krondorfer Christopher Shelswell Benjamin Huber-Dittes Dietmar Haltrich Clemens K Peterbauer
author_facet	Dagmar Brugger Iris Krondorfer Christopher Shelswell Benjamin Huber-Dittes Dietmar Haltrich Clemens K Peterbauer
author_sort	Dagmar Brugger
title	Engineering pyranose 2-oxidase for modified oxygen reactivity.
title_short	Engineering pyranose 2-oxidase for modified oxygen reactivity.
title_full	Engineering pyranose 2-oxidase for modified oxygen reactivity.
title_fullStr	Engineering pyranose 2-oxidase for modified oxygen reactivity.
title_full_unstemmed	Engineering pyranose 2-oxidase for modified oxygen reactivity.
title_sort	engineering pyranose 2-oxidase for modified oxygen reactivity.
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/5fffd99c82aa4bbcad797df5e930b929
work_keys_str_mv	AT dagmarbrugger engineeringpyranose2oxidaseformodifiedoxygenreactivity AT iriskrondorfer engineeringpyranose2oxidaseformodifiedoxygenreactivity AT christophershelswell engineeringpyranose2oxidaseformodifiedoxygenreactivity AT benjaminhuberdittes engineeringpyranose2oxidaseformodifiedoxygenreactivity AT dietmarhaltrich engineeringpyranose2oxidaseformodifiedoxygenreactivity AT clemenskpeterbauer engineeringpyranose2oxidaseformodifiedoxygenreactivity
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Engineering pyranose 2-oxidase for modified oxygen reactivity.

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