Coupled binuclear copper (CBC) enzymes

The coupled binuclear copper (CBC) protein family consists mainly of four subclasses, hemocyanins, tyrosinases, catechol oxidases and enzymes with hydroxyanilinase activity, which were recently added to this protein family. All CBC proteins are characterized by a coupled binuclear copper active site that reversibly binds O 2 in a symmetric side-on (-2: 2 ) fashion. Thus, the main function of hemocyanins is the oxygen transport in arthropods and mollusks, whereas catechol oxidases and tyrosinases catalyze the oxidation of diphenols to the corresponding quinones (diphenolase activity), whereby the latter enzyme is additionally able to ortho-hydroxylize monophenols (monophenolase activity). The fourth subclass, which includes the enzymes NspF and GriF, converts o-aminophenols into the corresponding o- nitrosophenol (hydroxyanilinaseactivity). While the protein structures of some representatives of the first three subclasses have already been solved, those of NspF and GriF are still unknown, however, their membership of the CBC protein family was proofed by spectroscopic and mutagenesis studies indicating that they possess a similar active site region. Thus, all four family members, despite exhibiting different catalytic reactions and substrate specificity, have a very similar active site architecture that fails to explain the difference in their activities. The main goal of this project is the crystallization and structure elucidation of NspF and GriF in order to find unique structural features that might explain their hydroxyanilinase activity and also why other members of this family are not abl e to catalyze this reaction. In addition, activity assays of NspF, GriF and tyrosinase (i.e. mushroom tyrosinase) with an array of substrates will be performed to determine the substrate specificity of each enzyme. Based on the kinetic and structural data decisive amino acid residues will be identified and mutated in order to verify their importance for a given activity. Thus, the results of this project will significantly contribute to the elucidation of the catalytic mechanism of CBC enzymes and might give answer to the following long -standing question: What factors give rise to the varied activities and/or substrate specificity among these oxidase classes? The gained knowledge of this project can then be used to optimize the applications of these enzymes, e.g. engineering a CBC enzyme that produces important intermediate molecules that can be used as lead structures for the synthesis of new drugs.