Tyrosinases and catechol oxidases are members of the class of type III copper enzymes. While tyrosinases accept both mono- and o-diphenols as substrates, only the latter substrate is converted by catechol oxidases. Researchers have been working for decades to elucidate the monophenolase/diphenolase specificity on a structural level and have introduced an early hypothesis that states that the reason for the lack of monophenolase activity in catechol oxidases may be its structurally restricted active site. However, recent structural and biochemical studies of this enzyme class have raised doubts about this theory. Herein, the first crystal structure of a plant tyrosinase (from Juglans regia) is presented. The structure reveals that the distinction between mono- and diphenolase activity does not depend on the degree of restriction of the active site, and thus a more important role for amino acid residues located at the entrance to and in the second shell of the active site is proposed.

Graphical abstractTyrosinase from walnut leaves (Juglans regia) corresponding to the known jrPPO1 sequence was purified and characterized. Two major tyrosinase forms differing only in their C-termini were identified. The first form (jrPPO1(Asp101 → Pro444)) is one amino acid shorter than the second form (jrPPO1(Asp101 → Arg445)).

Tyrosinase is a type 3 copper enzyme that catalyzes the ortho-hydroxylation of monophenols to diphenols as well as their subsequent oxidation to quinones, which are precursors for the biosynthesis of melanins. The first plant tyrosinase from walnut leaves (Juglans regia) was purified to homogeneity and crystallized. , and diffracted to 2.39 Å resolution. Crystals were only obtained from solutions containing at least 30% polyethylene glycol 5000 monomethyl ether in a close-to-neutral pH range.

Tyrosinasen und Catecholoxidasen gehçren zur Klasse der Typ-III-Kupferenzyme.F orscher arbeiten seit Jahrzehnten daran, die Monophenolase-/Diphenolase-Spezifität dieser Enzyme auf struktureller Ebene zu erklären und entwickelten dazu eine Hypothese (basierend auf der ersten Catechol-und Tyrosinasestruktur), der zufolge der Grund für das Fehlen von Monophenolase-Aktivität in Catecholoxidasen das strukturell eingeschränkte aktive Zentrum sein kçnnte. Jüngste strukturelle und biochemische Studien an dieser Enzymklasse liefern jedochAnlass zu Zweifeln an dieser Theorie. Hier wird die erste Kristallstruktur einer pflanzlichen Tyrosinase (Juglans regia) präsentiert, anhand derer gezeigt wird, dass die Unterscheidung zwischen Mono-und Diphenolase-Aktivität auf struktureller Ebene nicht durch eine strukturelle Einschränkung des aktiven Zentrums bestimmt wird;vielmehr spielen Aminosäuren, die sich am Eingang zu und innerhalb der zweiten Schale um das aktive Zentrum befinden, eine viel wichtigere Rolle,a ls bisher angenommen.

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