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Project 8

Crystallization and Characterization of the Type 3-Copper Protein Tyrosinase from Agaricus bisporus

Supervisor: Prof. Annette Rompel

Student: Stephan Mauracher

The enzyme tyrosinase is ubiquitous among a wide range of organisms from bacteria to fungi [1], plant and also mammals. The enzyme contains a type III copper center as an active site and catalyzes the two inducing reactions for the formation of the brown colored pigments (e.g. melanin).These pigments significantly lower disposal values of agricultural products [1].  For this reason food industry has a strong interest to generate structural and functional knowledge of the enzyme in order to establish appropriate ways to hinder its activity. The protein occurs in a latent precursor form which is activated, most likely due to proteolytic cleavage, to the active form, induced by cell damage and exposure to air.  Recently, the first crystal structure of an eukaryotic (Agaricus bisporus) active form of the enzyme was published [2].  For several decades now great effort for screening and developing an appropriate inhibitor for the active enzyme in terms of its functionality, availability and expenses, had been attempted. As a matter of fact no reasonable output was established. For this concern the more feasible approach might be the hindering of the activation process itself. Therefore it is necessary to elucidate the latent forms crystal structures.

The aim of this project is to isolate and purify tyrosinase of common edible mushroom (Agaricus bisporus) in reasonable quantity and near homogeneity in order to characterize its structure, most notable due to crystallization and X-ray diffraction structure analyses in its active and latent form. Moreover the rendering of assertions concerning the activation process (e.g. cleavage sites, responsible proteinases, binding interfaces, etc.) will be a task.

[1] S.Y. Seo, V. K. Sharma, N. Sharma: Mushroom Tyrosinase: Recent Prospects. Agric. Food Chem., 2003, 51 (10), 2837-2853.
[2] W.T. Ismaya , H.J. Rozeboom , A.  Weijn, J.J .Mes, F. Fusetti , H.J. Wichers , B.W. Dijkstra: Crystal structure of Agaricus bisporus mushroom tyrosinase: identity of the tetramer subunits and interaction with tropolone.
Biochem. 2011, 50 (24), 5477-5486.

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