Biochemical and structural characterisation of the copper containing oxidoreductases catechol oxidase, tyrosinase, and laccase from ascomycete fungi

Catechol oxidase (EC 1.10.3.1), tyrosinase (EC 1.14.18.1), and laccase (EC 1.10.3.2) are copper-containing metalloenzymes. They oxidise substituted phenols and use molecular oxygen as a terminal electron acceptor. Catechol oxidases and tyrosinases catalyse the oxidation of p-substituted o-diphenols to the corresponding o-quinones. Tyrosinases also catalyse the introduction of a hydroxyl group in the ortho position of p-substituted monophenols and the subsequent oxidation to the corresponding o-quinones. Laccases can oxidise a wide range of compounds by removing single electrons from the reducing group of the substrate and generate free radicals. The reaction products of these oxidases can react further non-enzymatically and lead to formation of polymers and crosslinking of proteins and carbohydrates, in certain conditions. The work focused on examination of the properties of catechol oxidases, tyrosinases and laccases. A novel catechol oxidase from the ascomycete fungus Aspergillus oryzae was characterised from biochemical and structural point of view. Tyrosinases from Trichoderma reesei and Agaricus bisporus were examined in terms of substrate specificity and inhibition. The oxidation capacity of laccases was elucidated by using a set of laccases with different redox potential and a set of substituted phenolic substrates with different redox potential. Finally, an evaluation of the protein cross-linking ability of catechol oxidase from A. oryzae, tyrosinases from T. reesei and A. bisporus and laccases from Trametes hirsuta, Thielavia arenaria, and Melanocarpus albomyces was performed. A novel extracellular catechol oxidase from A. oryzae (AoCO4; UniProtKB: Q2UNF9; Entrez gene ID: 5990879) was chosen for cloning and expression as representative of the newly discovered family of short tyrosinases sequences. AoCO4 gene was heterologously expressed in T. reesei. The protein produced did not show activity on L-tyrosine and 3,4-dihydroxy-L-phenylalanine (L-DOPA), which are typical substrates for tyrosinases. Consequently, the protein was classified as a catechol oxidase. AoCO4 was produced in a bioreactor and the expression resulted in high yields. The purified AoCO4 was partially processed at a Kex2/furin-type protease site and showed a molecular weight of 39.3 kDa. AoCO4 was able to oxidise a limited range of diphenolic compounds, e.g., catechol, caffeic acid, hydrocaffeic acid, and 4-tert-buthylcatechol. AoCO4 oxidised also the monophenolic compounds aminophenol and guaiacol. AoCO4 showed a pH optimum in the acidic range and was observed to be a relatively thermostable enzyme. A crystal structure of AoCO4 was solved at 2.5 Å