Catechol Oxidase versus Tyrosinase Classification Revisited by Site‐Directed Mutagenesis Studies

Catechol oxidases (COs) and tyrosinases (TYRs) are both polyphenol oxidases (PPOs) that catalyze the oxidation of ortho‐diphenols to the corresponding quinones. By the official classification, only TYRs can also catalyze the hydroxylation of monophenols to ortho‐diphenols. Researchers have been trying to find the molecular reason for the mono‐/diphenolase specificity for decades. However, the hypotheses for the lack of monophenolase activity of plant COs are only based on crystal structures so far. To test these hypotheses, we performed site‐directed mutagenesis studies and phylogenetic analyses with dandelion PPOs offering high phylogenetic diversity, the results of which refute the structure‐based hypotheses. While plant PPOs of phylogenetic group 2 solely exhibit diphenolase activity, plant PPOs of phylogenetic group 1 unexpectedly also show monophenolase activity. This finding sheds new light upon the molecular basis for mono‐/diphenol substrate specificity and challenges the current practice of generally naming plant PPOs as COs. Pusteblume! Die ortsgerichtete Mutagenese phylogenetisch diverser Polyphenoloxidasen (PPOs) aus dem Löwenzahn widerspricht der Kristallstruktur‐basierten Hypothese, dass pflanzliche PPOs exklusiv als Diphenolasen (Catecholoxidasen, COs) wirken. Pflanzliche PPOs der phylogenetischen Gruppe 2 sind COs, während solche der Gruppe 1 zusätzlich Monophenolaseaktivität zeigen und daher tatsächlich Tyrosinasen (TYRs) sind.