ИСТИНА

D-amino acid oxidase (DAAO, EC 1.4.3.3) catalyzes oxidation of D-amino acids with strict stereo specificity to corresponding alpha-ketoacids, ammonia and hydrogen peroxide. For nowadays, it is the enzyme of high scientific interest in terms of physiological role in living organisms and biotechnological application for production of optically pure L-amino acids and α-keto acids, creation of biosensors and for bioconversion of cephalosporin C to 7-aminocephalosporanic acid. Regarding industrial application, the most attractive enzyme is DAAO from the yeast Trigonopsis variabilis (TvDAAO). Unfortunately, properties of the enzyme are often non optimal for application. We explore TvDAAO with rational design with the aim to study structure functional relationships as well as optimization of properties for application, i.e. stability and catalytic activity. Modeling and solution of mutant TvDAAO structure provided background for our studies, which include computer analysis of enzyme’s structure, docking of substrates, and alignment of DAAOs sequences and simulation of mutations. Here we will present results of rational design of TvDAAO. Roles of Met104 and of the loop from 99 to 110 in aa sequence were studied. Loop 99 110 and comprising residues were essential for thermal stability and activity of TvDAAO. For instance, aromatic substitutions of Met104 dramatically increased thermal stability in contrast to small hydrophilic, hydrophobic and charged amino acids. Met104Phe had 11 times higher period of half inactivation at 56 0C in comparison to wild type. To figure out the nature of stabilization effect six double mutants as a combination of Met104Phe, Tyr, Trp with spatially closed Phe54Ser and Phe258Ser were obtained. Finally, Met104Phe led to drastic stabilization of TvDAAO was combined with mutations in FAD-binding domain and active site of the enzyme to give a series of multipoint mutants with improved catalytic activity and thermal stability.