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Site‐directed mutagenesis around the CuA site of a polyphenol oxidase from Coreopsis grandiflora ( cg AUS1)
Author(s) -
Kaintz Cornelia,
Mayer Rupert L.,
Jirsa Franz,
Halbwirth Heidi,
Rompel Annette
Publication year - 2015
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2015.02.009
Subject(s) - chemistry , polyphenol oxidase , alanine , site directed mutagenesis , mutagenesis , biochemistry , cysteine , active site , enzyme , stereochemistry , amino acid , mutation , peroxidase , mutant , gene
Aurone synthase from Coreopsis grandiflora ( cg AUS1), 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.