Site‐directed mutagenesis around the CuA site of a polyphenol oxidase from Coreopsis grandiflora (cgAUS1). Issue 7 (16th February 2015)

• Record Type: Journal Article
• Authors: Kaintz, Cornelia; Mayer, Rupert L.; Jirsa, Franz; Halbwirth, Heidi; Rompel, Annette
• Is Part Of: FEBS letters. Volume 589:Issue 7(2015)
• Page Start: 789
• Page End: 797
• Publication Date: 2015-02-16
• Subjects: AAS -- atomic absorption spectroscopy -- AmAS1 -- aureusidin synthase from Antirrhinum majus (protein) -- aoTYR -- tyrosinase from Aspergillus oryzae (protein) -- bmTYR -- tyrosinase from Bacillus megaterium (protein) -- cgAUS1 -- aurone synthase from Coreopsis grandiflora (protein) -- cgAUS1 -- aurone synthase from Coreopsis grandiflora (gene) -- F-AAS -- flame atomic absorption spectroscopy -- GF-AAS -- graphite furnace atomic absorption spectroscopy -- GST -- glutathione S-transferase -- ibCO -- catechol oxidase from Ipomoea batatas (protein) -- IPTG -- isopropyl-β-d-thiogalactopyranoside -- LC -- liquid chromatography -- LC/MS -- liquid chromatography / mass spectrometry -- nanoUHPLC–ESI-MS/MS -- ultra high performance liquid chromatography–electrospray tandem mass spectrometry -- PES -- poly(oxy-1, 4-phenylsulfonyl-1, 4-phenyl) -- PHC -- 2′, 3, 4, 4′, 6′-pentahydroxychalcone -- PMSF -- phenylmethylsulfonylfluorid -- PPO -- polyphenol oxidase -- PPO-6 -- dandelion PPO-6 from Taraxacum officinale (protein) -- TBC -- 4-tert-butylcatechol -- THC -- 2′, 4, 4′, 6′-tetrahydroxychalcone -- vvCO -- catechol oxidase from Vitis vinifera (protein) -- Type-3 copper protein -- Polyphenol oxidase (PPO) -- Aurone synthase (AUS) -- Site-directed mutagenesis -- 4-Deoxyaurone -- Copper binding site; Biochemistry -- Periodicals; Biophysics -- Periodicals; Molecular biology -- Periodicals; Biochimie -- Périodiques; Biochemistry; Biophysics; Molecular biology; Periodicals; 572.05
• DOI: 10.1016/j.febslet.2015.02.009
• Languages: English
• ISSNs: 0014-5793

Abstract

Abstract : 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. Abstract : Site‐directed mutations of AUS1 around the CuA site were generated and verified. All mutations led to loss of diphenolase activity with butein as substrate. Exchange of histidines in the CuA resulted in enzymes containing only one Cu. F273 mutation to alanine did not increase the monophenolase activity. C97 mutation eliminated the diphenolase activity, but 2 Cu atoms were incorporated.