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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.

SUBMITTER: Kaintz C

PROVIDER: S-EPMC4364613 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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Site-directed mutagenesis around the CuA site of a polyphenol oxidase from Coreopsis grandiflora (cgAUS1).

Kaintz Cornelia C Mayer Rupert L RL Jirsa Franz F Halbwirth Heidi H Rompel Annette A

FEBS letters 20150216 7

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 ...[more]

PMID: 25697959

Similar Datasets

Crystallization and preliminary crystallographic analysis of latent, active and recombinantly expressed aurone synthase, a polyphenol oxidase, from Coreopsis grandiflora.

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Cloning and functional expression in E. coli of a polyphenol oxidase transcript from Coreopsis grandiflora involved in aurone formation.

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Latent and active aurone synthase from petals of C. grandiflora: a polyphenol oxidase with unique characteristics.

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Project description:Multicopper oxidases can act on a broad spectrum of phenolic and non-phenolic compounds. These enzymes include laccases, which are widely distributed in plants and fungi, and were more recently identified in bacteria. Here, we present the results of biochemical and mutational studies of small laccase (SLAC), a multicopper oxidase from Streptomyces coelicolor (SCO6712). In addition to typical laccase substrates, SLAC was tested using phenolic compounds that exhibit antioxidant activity. SLAC showed oxidase activity against 12 of 23 substrates tested, including caffeic acid, ferulic acid, resveratrol, quercetin, morin, kaempferol and myricetin. The kinetic parameters of SLAC were determined for 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid), 2,6-dimethoxyphenol, quercetin, morin and myricetin, and maximum reaction rates were observed with myricetin, where kcat and Km values at 60°C were 8.1 (±?0.8) s?¹ and 0.9 (±?0.3) mM respectively. SLAC had a broad pH optimum for activity (between pH?4 and 8) and temperature optimum at 60-70°C. It demonstrated remarkable thermostability with a half-life of over 10?h at 80°C and over 7?h at 90°C. Site-directed mutagenesis revealed 17 amino acid residues important for SLAC activity including the 10 His residues involved in copper coordination. Most notably, the Y229A and Y230A mutant proteins showed over 10-fold increase in activity compared with the wild-type SLAC, which was correlated to higher copper incorporation, while kinetic analyses with S929A predicts localization of this residue near the meta-position of aromatic substrates.

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| S-EPMC4707547 | biostudies-literature

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