RESUMO
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.
Assuntos
Catecol Oxidase/genética , Cobre/metabolismo , Coreopsis/enzimologia , Mutagênese Sítio-Dirigida , Proteínas de Plantas/genética , Alanina/metabolismo , Sítios de Ligação , Catecol Oxidase/metabolismo , Chalconas/metabolismo , Coreopsis/genética , Cisteína/metabolismo , Proteínas de Plantas/metabolismoRESUMO
Recent investigations in the study of plant, fungal, and bacterial type-3 copper proteins are reviewed. Focus is given to three enzymes: catechol oxidases (CO), tyrosinases, and aureusidin synthase. CO were mostly found in plants, however, in 2010 the first fungal CO was published. The first plant-originated tyrosinase was published in 2014, before tyrosinases were only reported in fungi, bacteria, and human. Aureusidin synthase from yellow snapdragon (Antirrhinum majus) was first published in 2000, as part of yellow flower coloration pathway. In the last years, many important results on type-3 copper enzymes originated from X-ray crystallographic investigations. In addition, studies on site-directed mutagenesis of amino acids around the active site were performed to identify the regions determining monophenolase and/or diphenolase activity. Although X-ray crystallographic structures of CO and tyrosinases are available, many questions like the response for the activation via proteases, sequence-based or structural-based differences between CO, as well as the physiological roles of many polyphenol oxidases still remain to be addressed.
Assuntos
Proteínas de Bactérias/química , Catecol Oxidase/química , Cobre/química , Proteínas Fúngicas/química , Oxigenases de Função Mista/química , Monofenol Mono-Oxigenase/química , Proteínas de Plantas/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Domínio Catalítico , Catecol Oxidase/genética , Cristalografia por Raios X , Proteínas Fúngicas/genética , Humanos , Oxigenases de Função Mista/genética , Modelos Moleculares , Dados de Sequência Molecular , Monofenol Mono-Oxigenase/genética , Mutação , Proteínas de Plantas/genética , Alinhamento de Sequência , Relação Estrutura-AtividadeRESUMO
Polyphenol oxidases are involved in aurone biosynthesis but the gene responsible for 4-deoxyaurone formation in Asteraceae was so far unknown. Three novel full-length cDNA sequences were isolated from Coreopsis grandiflora with sizes of 1.80kb (cgAUS1) and 1.85kb (cgAUS2a, 2b), encoding for proteins of 68-69kDa, respectively. cgAUS1 is preferably expressed in young petals indicating a specific role in pigment formation. The 58.9kDa AUS1 holoproenzyme, was recombinantly expressed in E. coli and purified to homogeneity. The enzyme shows only diphenolase activity, catalyzing the conversion of chalcones to aurones and was characterized by SDS-PAGE and shot-gun type nanoUHPLC-ESI-MS/MS.