Tyrosinase is responsible for the two initial enzymatic steps in the conversion of tyrosine to melanin. Many tyrosinase mutations are the leading cause of albinism in humans, and it is a prominent biotechnology and pharmaceutical industry target. Here we present crystal structures that show 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. Overall, the structures reveal precise steps in the enzymatic catalytic cycle as well as differences between tyrosinases and other type-3 copper enzymes.

Tyrosinases are metalloenzymes belonging to the type-3 copper protein family which contain two copper ions in the active site. They are found in various prokaryotes as well as in plants, fungi, arthropods, and mammals and are responsible for pigmentation, wound healing, radiation protection, and primary immune response. Tyrosinases perform two sequential enzymatic reactions: hydroxylation of monophenols and oxidation of diphenols to form quinones which polymerize spontaneously to melanin. Two other members of this family are catechol oxidases, which are prevalent mainly in plants and perform only the second oxidation step, and hemocyanins, which lack enzymatic activity and are oxygen carriers. In the last decade, several structures of plant and bacterial tyrosinases were determined, some with substrates or inhibitors, highlighting features and residues which are important for copper uptake and catalysis. This review summarizes the updated information on structure-function correlations in tyrosinases along with comparison to other type-3 copper proteins.

Tyrosinases are responsible for melanin formation in all life domains. Tyrosinase inhibitors are used for the prevention of severe skin diseases, in skin-whitening creams and to avoid fruit browning, however continued use of many such inhibitors is considered unsafe. In this study we provide conclusive evidence of the inhibition mechanism of two well studied tyrosinase inhibitors, KA (kojic acid) and HQ (hydroquinone), which are extensively used in hyperpigmentation treatment. KA is reported in the literature with contradicting inhibition mechanisms, while HQ is described as both a tyrosinase inhibitor and a substrate. By visualization of KA and HQ in the active site of TyrBm crystals, together with molecular modeling, binding constant analysis and kinetic experiments, we have elucidated their mechanisms of inhibition, which was ambiguous for both inhibitors. We confirm that while KA acts as a mixed inhibitor, HQ can act both as a TyrBm substrate and as an inhibitor.

Protocells, artificial cell-like particles, capable of autonomously synthesizing RNA and proteins based on a DNA template, are emerging platforms for studying cellular functions and for revealing the origins-of-life. Here, we show for the first time that artificial lipid-based vesicles, containing the molecular machinery necessary for transcription and translation, can be used to synthesize anti-cancer proteins inside tumors. The particles were engineered as stand-alone systems, sourcing nutrients from their biological microenvironment to trigger protein synthesis. When pre-loaded with template DNA, amino acids and energy-supplying molecules protocells synthesized, up to 2×107 copies of superfolder green fluorescent protein (sfGFP) were synthesized in each liposome. A variety of proteins, having molecular weights reaching 66 kDa and with diagnostic and therapeutic activities, were synthesized inside the particles. Incubating protein producing particles, encoded to secrete Pseudomonas exotoxin A (PE) with 4T1 breast cancer cells in culture, resulted in killing of most of the malignant cells. In mice bearing 4T1 tumors, histological evaluation of the tumor tissue after a local injection of PE-producing particles, indicating robust apoptosis. Protein producing particles are synthetic-biology platforms capable of synthesizing therapeutic proteins on-demand.

Personalized medicine promises to revolutionize cancer therapy by matching the most effective treatment to the individual patient. Using a nanoparticle-based system, we predict the therapeutic potency of anticancer medicines in a personalized manner. We carry out the diagnostic stage through a multidrug screen performed inside the tumour, extracting drug activity information with single cell sensitivity. By using 100 nm liposomes, loaded with various cancer drugs and corresponding synthetic DNA barcodes, we find a correlation between the cell viability and the drug it was exposed to, according to the matching barcodes. Based on this screen, we devise a treatment protocol for mice bearing triple-negative breast-cancer tumours, and its results confirm the diagnostic prediction. We show that the use of nanotechnology in cancer care is effective for generating personalized treatment protocols.