title: Site-directed mutagenesis around the CuA site of a polyphenol oxidase from Coreopsis grandiflora (cgAUS1).
abstract: Aurone synthase from Coreopsis grandiflora (cgAUS1), catalyzing conversion of butein to sulfuretin in a type-3 copper center, is a rare example of a polyphenol oxidase involved in anabolism. Site-directed mutagenesis around the CuA site of AUS1 was performed, and recombinant enzymes were analyzed by mass spectrometry. Replacement of the coordinating CuA histidines with alanine resulted in the presence of a single copper and loss of diphenolase activity. The thioether bridge-building cysteine and a phenylalanine over the CuA site, exchanged to alanine, have no influence on copper content but appear to play an important role in substrate binding.
authors.affiliation: Universität Wien, Fakultät für Chemie, Institut für Biophysikalische Chemie, Althanstraße 14, 1090 Wien, Austria. Electronic address: cornelia.kaintz@univie.ac.at.
authors.forename: Cornelia
authors.initials: C
authors.lastname: Kaintz
authors.affiliation: Universität Wien, Department of Analytical Chemistry, Währinger Straße 38, 1090 Vienna, Austria. Electronic address: rupert.mayer@univie.ac.at.
authors.forename: Rupert L
authors.initials: RL
authors.lastname: Mayer
authors.affiliation: Universität Wien, Department of Inorganic Chemistry, Althanstraße 14, 1090 Vienna, Austria. Electronic address: franz.jirsa@univie.ac.at.
authors.forename: Franz
authors.initials: F
authors.lastname: Jirsa
authors.affiliation: University of Technology Vienna, Institute of Chemical Engineering, Getreidemarkt 9, 1060 Vienna, Austria. Electronic address: hhalb@mail.zserv.tuwien.ac.at.
authors.forename: Heidi
authors.initials: H
authors.lastname: Halbwirth
authors.affiliation: Universität Wien, Fakultät für Chemie, Institut für Biophysikalische Chemie, Althanstraße 14, 1090 Wien, Austria. Electronic address: annette.rompel@univie.ac.at.
authors.forename: Annette
authors.initials: A
authors.lastname: Rompel
mesh_terms: D000409:Alanine / Q000378:metabolism; D001665:Binding Sites; D004156:Catechol Oxidase / Q000235:genetics* / Q000378:metabolism; D047188:Chalcones / Q000378:metabolism; D003300:Copper / Q000378:metabolism*; D031945:Coreopsis / Q000201:enzymology* / Q000235:genetics; D003545:Cysteine / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D047188:Chalcones; D010940:Plant Proteins; C040918:butein; D003300:Copper; D004156:Catechol Oxidase; D003545:Cysteine; D000409:Alanine
keywords: 4-Deoxyaurone; Aurone synthase (AUS); Copper binding site; Polyphenol oxidase (PPO); Site-directed mutagenesis; Type-3 copper protein