ABSTRACT
Human tyrosinase is the first enzyme of the multistep process of melanogenesis. It catalyzes the hydroxylation of L-tyrosine to L-dihydroxyphenylalanine and the following oxidation of o-diphenol to the corresponding quinone, L-dopaquinone. In spite of its biomedical relevance, its reactivity is far from being fully understood, mostly because of the lack of a suitable expression system. Indeed, until now, studies on substrates and inhibitors of tyrosinases have been performed in vitro almost exclusively using mushroom or bacterial enzymes. We report on the production of a recombinant human tyrosinase in insect cells (Sf9 line). Engineering the protein, improving cell culture conditions, and setting a suitable purification protocol optimized product yield. The obtained active enzyme was truthfully characterized with a number of substrate and inhibitor molecules. These results were compared to those gained from a parallel analysis of the bacterial (Streptomyces antibioticus) enzyme and those acquired from the literature for mushroom tyrosinase, showing that the reactivity of the human enzyme appears unique and pointing out the great bias introduced when using non-human tyrosinases to measure the inhibitory efficacy of new molecules. The described enzyme is therefore an indispensable paradigm in testing pharmaceutical or cosmetic agents addressing tyrosinase activity.
Subject(s)
Drug Evaluation, Preclinical , Insecta/metabolism , Monophenol Monooxygenase/metabolism , Agaricales/enzymology , Animals , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , Kinetics , Monophenol Monooxygenase/antagonists & inhibitors , Mutant Proteins/metabolism , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sf9 Cells , Streptomyces/enzymology , Viruses/metabolismABSTRACT
Diabetes shows a wide range of variation in prevalence around the world and it is expected to affect 300 million by the year 2025. In a prevention framework where banning policies and educational strategies lead the interventions, functional foods (FFs) with their specific health effects could, in the future, indicate a new mode of thinking about the relationships between food and health in everyday life. Functional ingredients, such as stevioside, cinnamon, bitter melon, garlic and onion, ginseng, Gymnema sylvestre and fenugreek, have been addressed for their specific actions towards different reactions involved in diabetes development. New strategies involving the use of FF should be validated through large-scale population trials, considering validated surrogate end points to evaluate the effect of FF in prevention of chronic diseases such as type 2 diabetes mellitus.
