| Abstract [eng] |
The two main objects of this master’s thesis are enzymes Vcz and KANOS – deaminases from the metagenomic libraries, discovered at the Department of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Vilnius University. It is a well-known fact that the chemotherapeutic drug 5-fluorouracil can be a product of a deaminase-catalyzed reaction. Hence, it was decided to investigate the applicability of Vcz and KANOS deaminases for targeted cancer therapy. In vitro enzyme evolution methods were used to improve the kinetic parameters of Vcz deaminase using isocytosine as its substrate. The effectiveness of the methods applied has been demonstrated, but due to the lack of a proper mutant enzyme selection system, the improved variant of Vcz deaminase could not be obtained. The ability of KANOS deaminase to catalyze the deamination of both cytosine and isocytosine was compared with that of a typical cytosine deaminase CodA from E. coli. The kinetic parameters of KANOS for isocytosine deamination were ~1.5 times higher than those of CodA, suggesting that this enzyme is suitable for further application in targeted cancer therapy. Also, a hybrid system that uses a two-enzyme (Vcz deaminase and D8 amidohydrolase) hybrid and a single prodrug (5-fluoro-N2-acetylisocytosine) is proposed. It was shown in vivo that uracil can be obtained from N2-acetylisocytosine in a two-step reaction, using both Vcz deaminase and D8 amidohydrolase in a hybrid enzyme. Using complex prodrugs that need several enzymatic steps to be converted into the active anti-cancer drug (5-fluorouracil) should reduce the severity of the side effects that accompany enzyme-prodrug cancer therapies such as cytosine deaminase / 5-fluorocytosine in their clinical application. |
