Tyrosinase is a di-copper oxidase responsible for the production of melanin in many organisms. It is involved in wound healing, UV protection, detoxification of phenols and more. Mutations in tyrosinase cause albinism in mammals and are associated with melanoma. The active site of all type-3-copper proteins is identical among tyrosinases, catechol oxidases, and hemocyanins, yet each member of the family has different functions. The catalytic mechanism of type-3-copper proteins has been studied extensively for the past two decades, however, to date, these studies have not provided direct evidence of the catalytic cycle, as visualization of substrates in the active site has not been obtained.
In this study, we present crystal structures that show unequivocally that both monophenol hydroxylation and diphenol oxidation occur at the same site. It is suggested that concurrent presence of a phenylalanine above the active site and a restricting thioether bond on the histidine coordinating CuA prevent hydroxylation of monophenols by catechol oxidases. Furthermore, a conserved water molecule activated by E195 and N205, is proposed to mediate deprotonation of the monophenol at the active site. The new structures reveal precise steps in the enzymatic catalytic cycle as well as differences between tyrosinases and other type-3 copper enzymes. This work describing substrate orientation in the active site, together with the ability to crystalize tyrosinase with mostly any substrate, now paves the way for designing new tyrosinase inhibitors for food, pharmaceutical, and cosmetic applications.
