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Abstract
Tyrosinase is a copper-binding enzyme involved in melanin biosynthesis. However, the detailed structure of human tyrosinase has not yet been solved, along with the identification of the key sites responsible for its catalytic activity. We used site-directed mutagenesis to identify the residues critical for the copper binding of human tyrosinase. Seven histidine mutants in the two copper-binding sites were generated, and catalytic activities were characterised. The tyrosine hydroxylase activities of the CuA site mutants were approximately 50% lower than those of the wild-type tyrosinase, while the dopa oxidation activities of the mutants were not significantly different from that of wild-type tyrosinase. By contrast, mutations at CuB significantly decreased both tyrosine hydroxylation and dopa oxidation activities, confirming that the catalytic sites for these two activities are at least partially distinct. These findings provide a useful resource for further structural determination and development of tyrosinase inhibitors in the cosmetic and pharmaceutical industries.
Highlights
Melanin biosynthesis is a complicated pathway involving chemical and enzymatic reactions and is limited to melanocytes in mammals
A high yield of tyrosinase expression was obtained using E. coli BL21 Star (DE3) cells followed by purification, which was greater than that obtained in our previous study[24], demonstrating the efficiency of the 6Â His tag at the C-terminal rather than the N-terminal
Recombinant wild-type and mutant human tyrosinase enzymes were overexpressed in E. coli BL21 (DE3) and utilised to analyse the catalytic activity
Summary
Melanin biosynthesis is a complicated pathway involving chemical and enzymatic reactions and is limited to melanocytes in mammals. Tyrosinase (monophenol monooxygenase, EC 1.14.18.1) plays a pivotal role in the melanin synthesis pathway. Tyrosinase is the only human melanogenic enzyme with well-established in vivo catalytic enzyme activity[1], catalysing several steps in melanin synthesis and generated by the hydroxylation of L-tyrosine[2,3,4]. Tyrosinase is a copper-containing metalloprotein belonging to the type-3 copper protein family, together with haemocyanins and catechol oxidases. These proteins are abundant in mammals, bacteria, fungi, and plants, and the active sites are highly conserved among the different species[5]. Human tyrosinase is a quite attractive target for medical and industrial applications. The screening of potent antagonists of tyrosinase and their subsequent development to drugs have attracted substantial interest in the cosmetic industry
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