Engineering of pyranose dehydrogenase for increased oxygen reactivity.

Pyranose dehydrogenase (PDH), a member of the GMC family of flavoproteins, shows a very broad sugar substrate specificity but is limited to a narrow range of electron acceptors and reacts extremely slowly with dioxygen as acceptor. The use of substituted quinones or (organo)metals as electron accept...

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Autores principales:	Iris Krondorfer, Katharina Lipp, Dagmar Brugger, Petra Staudigl, Christoph Sygmund, 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/5900f04c2c2548ffa3ba856c3907b1fa
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spelling	oai:doaj.org-article:5900f04c2c2548ffa3ba856c3907b1fa2021-11-18T08:28:50ZEngineering of pyranose dehydrogenase for increased oxygen reactivity.1932-620310.1371/journal.pone.0091145https://doaj.org/article/5900f04c2c2548ffa3ba856c3907b1fa2014-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24614932/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203Pyranose dehydrogenase (PDH), a member of the GMC family of flavoproteins, shows a very broad sugar substrate specificity but is limited to a narrow range of electron acceptors and reacts extremely slowly with dioxygen as acceptor. The use of substituted quinones or (organo)metals as electron acceptors is undesirable for many production processes, especially of food ingredients. To improve the oxygen reactivity, site-saturation mutagenesis libraries of twelve amino acids around the active site of Agaricus meleagris PDH were expressed in Saccharomyces cerevisiae. We established high-throughput screening assays for oxygen reactivity and standard dehydrogenase activity using an indirect Amplex Red/horseradish peroxidase and a DCIP/D-glucose based approach. The low number of active clones confirmed the catalytic role of H512 and H556. Only one position was found to display increased oxygen reactivity. Histidine 103, carrying the covalently linked FAD cofactor in the wild-type, was substituted by tyrosine, phenylalanine, tryptophan and methionine. Variant H103Y was produced in Pichia pastoris and characterized and revealed a five-fold increase of the oxygen reactivity.Iris KrondorferKatharina LippDagmar BruggerPetra StaudiglChristoph SygmundDietmar HaltrichClemens K PeterbauerPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 3, p e91145 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Iris Krondorfer Katharina Lipp Dagmar Brugger Petra Staudigl Christoph Sygmund Dietmar Haltrich Clemens K Peterbauer Engineering of pyranose dehydrogenase for increased oxygen reactivity.
description	Pyranose dehydrogenase (PDH), a member of the GMC family of flavoproteins, shows a very broad sugar substrate specificity but is limited to a narrow range of electron acceptors and reacts extremely slowly with dioxygen as acceptor. The use of substituted quinones or (organo)metals as electron acceptors is undesirable for many production processes, especially of food ingredients. To improve the oxygen reactivity, site-saturation mutagenesis libraries of twelve amino acids around the active site of Agaricus meleagris PDH were expressed in Saccharomyces cerevisiae. We established high-throughput screening assays for oxygen reactivity and standard dehydrogenase activity using an indirect Amplex Red/horseradish peroxidase and a DCIP/D-glucose based approach. The low number of active clones confirmed the catalytic role of H512 and H556. Only one position was found to display increased oxygen reactivity. Histidine 103, carrying the covalently linked FAD cofactor in the wild-type, was substituted by tyrosine, phenylalanine, tryptophan and methionine. Variant H103Y was produced in Pichia pastoris and characterized and revealed a five-fold increase of the oxygen reactivity.
format	article
author	Iris Krondorfer Katharina Lipp Dagmar Brugger Petra Staudigl Christoph Sygmund Dietmar Haltrich Clemens K Peterbauer
author_facet	Iris Krondorfer Katharina Lipp Dagmar Brugger Petra Staudigl Christoph Sygmund Dietmar Haltrich Clemens K Peterbauer
author_sort	Iris Krondorfer
title	Engineering of pyranose dehydrogenase for increased oxygen reactivity.
title_short	Engineering of pyranose dehydrogenase for increased oxygen reactivity.
title_full	Engineering of pyranose dehydrogenase for increased oxygen reactivity.
title_fullStr	Engineering of pyranose dehydrogenase for increased oxygen reactivity.
title_full_unstemmed	Engineering of pyranose dehydrogenase for increased oxygen reactivity.
title_sort	engineering of pyranose dehydrogenase for increased oxygen reactivity.
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/5900f04c2c2548ffa3ba856c3907b1fa
work_keys_str_mv	AT iriskrondorfer engineeringofpyranosedehydrogenaseforincreasedoxygenreactivity AT katharinalipp engineeringofpyranosedehydrogenaseforincreasedoxygenreactivity AT dagmarbrugger engineeringofpyranosedehydrogenaseforincreasedoxygenreactivity AT petrastaudigl engineeringofpyranosedehydrogenaseforincreasedoxygenreactivity AT christophsygmund engineeringofpyranosedehydrogenaseforincreasedoxygenreactivity AT dietmarhaltrich engineeringofpyranosedehydrogenaseforincreasedoxygenreactivity AT clemenskpeterbauer engineeringofpyranosedehydrogenaseforincreasedoxygenreactivity
_version_	1718421741351469056

Engineering of pyranose dehydrogenase for increased oxygen reactivity.

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