טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentDeri Zenaty Batel
SubjectStructure-Function Studies of Bacterial and Mammalian
Tyrosinase and their Inhibitors
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Ayelet Fishman
Full Thesis textFull thesis text - English Version


Abstract

Tyrosinases are type-3 copper-containing enzymes responsible for the formation of melanin. Disorder in melanin formation has been linked to a variety of skin diseases in humans, thus tyrosinase inhibitors are desired for their prevention. The main goal of this research was to study the mode of tyrosinase inhibition and to develop new and safe inhibitors. The project focused on various inhibitors: (i) skin whitening products as kojic acid (KA) and hydroquinone (HQ), (ii) novel small molecule inhibitors, and (iii) novel peptide-based inhibitors. Tyrosinase from Bacillus megaterium (TyrBm) served as the main model enzyme in this study.

Regarding the commercially available inhibitors, KA was characterized as a mixed inhibitor, while HQ could act both as a tyrosinase substrate and as an inhibitor. Crystal structures of TyrBm were determined with different conformations of bound KA and HQ in the active site.

A novel series of indole derivatives was designed for the inhibition of tyrosinase with compound 4-(4-fluorobenzyl)piperazin-1-yl](2,4-dinitrophenyl)methanone (IC50 value of 0.96 μM) being 20-fold more potent than the positive control KA. It was also proved to exhibit no considerable cytotoxic effect in B16F10 melanoma cells. Based on the X-ray crystal structure, this compound is able to bind with the 4-fluorobenzyl moiety towards the copper ions at the catalytic site of tyrosinase.

The crystal structure of TyrBm with a new bound inhibitor, peptide VSHYDPVP, was determined at 2.5 Å resolution. The binding of the peptide caused a movement of crucial residues Arg209, Asn205 and His60 as well as polar interactions with Arg209 and Glu195, enabling its stabilization.

In order to design novel inhibitors with specific binding to the active site of the human tyrosinase we attempted to obtain a crystal structure which is not available to date. A truncated version of the WT enzyme and a quadruple mutant with less glycosylation sites, were constructed, expressed in mammalian cells, and purified. The use of in vivo glycosylation inhibitors and post enzymatic deglycosylation did not yield a homogeneous protein solution for crystallization.

An additional aim was to develop a new platform for the synthesis of the antioxidant hydroxytyrosol (HT) using tyrosinase as the biocatalyst. For an efficient production of HT, we developed a continuous two-enzyme reaction system using sol-gel immobilized tyrosinase and glucose dehydrogenase (GDH). While tyrosinase oxidized tyrosol to 3,4-quinone-phenylethanol, GDH catalyzed the simultaneous reduction of NAD to NADH, which was the reducing agent enabling the accumulation of HT from the quinone. Using 50 mM tyrosol, the immobilized system under optimized conditions, enabled a final HT yield of 7.68 g/L with productivity of 2.30 mg HT/mg TyrBm beads.

To sum, this work mostly explored the inhibition of tyrosinases from different domains of life, and demonstrated new motifs and challenges in designing novel inhibitor compounds. However, there is still a need in compounds oriented specifically towards the binding pocket of the human enzyme. Furthermore, the use of tyrosinase in biocatalysis was also investigated and revealed a beneficial system for the efficient synthesis of diphenols.