Journal:JBIC:22

The crystal structure of an extracellular catechol oxidase from the ascomycete fungus Aspergillus oryzae

Nina Hakulinen�, Chiara Gasparetti, Heidi Kaljunen, Kristiina Kruus, and Juha Rouvinen ^[1]

Molecular Tour
Catechol oxidases (EC 1.10.3.1) catalyse the oxidation of o-diphenols to their corresponding o-quinones. These oxidases contain two copper ions (CuA and CuB) within the so-called coupled binuclear type-3 copper site as found in tyrosinases (EC 1.14.18.1) and haemocyanins. We determined the first crystal structure of a fungal catechol oxidase from Aspergillus oryzae (AoCO4).

Two different forms of AoCO4, called as a full-length and a truncated, were crystallized and the structures were solved at 2.5 and 2.9 ֵ resolution, respectively. The overall structure of AoCO4 is predominantly α-helical. A four-helix bundle forms the core of the protein and the catalytic copper site is situated within this helical bundle. The truncated form lacks the long N-terminal α-helix (shown in cyan), which is not part of the central helical bundle (shown in green). Both the full-length and truncated form exists as a similar transient dimer (surface area 855 ֵ2) in crystal.

The crystal structure of AoCO4 demonstrated that mono-oxygenase and diphenolase reactivity cannot be explained by accessibility to copper ions. Based on the observations that CuA is restricted by a Phe residue in plant catechol oxidases, but not in tyrosinases, it has been suggested that o-diphenols bind to CuB, whereas monophenols bind to CuA. However, both copper ions were solvent-exposed and accessible to substrates in AoCO4.

The crystal structure of the full-length AoCO4 revealed an elongated electron density between CuA and CuB in the catalytic centre. This was best refined as a diatomic oxygen moiety. The O2 atom of the dioxygen moiety was approximately 2.0 ֵ and 2.3 ֵ away from CuA and CuB, respectively, and the O1 atom of dioxygen moiety was 2.6 ֵ away from each copper ion. Furthermore, the UV/VIS absorption spectrum indicated that enzyme exists partially in the oxy-form, because native form as isolated exhibited a clear absorption band at 350 nm.