ИСТИНА

D-amino acid oxidase (DAAO, EC 1.4.3.3) is a well-known FAD-containing oxidoreductase that catalyzes oxygen dependent oxidation of D-amino acids with strict stereo specificity to corresponding imino acids, ammonia and hydrogen peroxide [1]. DAAO was discovered by H.A.Krebs in 1935 during oxidation of L- and D-amino acids with porcine kidney and liver homogenates and it was the second found flavoenzyme [2]. During last 70 years DAAO has been the focus of many research and identified in wide range of organisms from yeasts and bacteria to human where it play essential roles [3]. For nowadays, it is still the enzyme of high scientific interest in terms of physiological role in human organism and biotechnological application for production of optically pure unnatural L-amino acids and α-keto acids, creation of biosensors and two-step enzymatic conversion of Cephalosporin C to 7-aminocephalosporanic acid [4,5]. Regarding industrial application the most attractive enzyme is DAAO from yeast Trigonopsis variabilis (TvDAAO). Gene of TvDAAO was cloned in our laboratory and expressed in E.coli as soluble and active enzyme. Modeling and solution of mutant TvDAAO structure provided background for studies of TvDAAO with rational design technique that includes computer analysis of TvDAAO’s structure, docking of substrates to the active site, alignment of DAAOs sequences and simulation of mutations. Currently our laboratory deals with the study of structure-functional relationship of TvDAAO as well as obtaining enzymes with improved properties in terms of thermal stability and catalytic activity. Here we will present the latest results on protein engendering of TvDAAO. As a part of structural design the roles of Met104 residue [6] and of the loop from 99 to 110 in amino acid sequence in thermal stability and catalytic properties were studied. The loop 99-110 was found to be essential for thermal stability of TvDAAO. Introducing of single point mutants in 104th position showed the correlation between shape, charge and hydrophobicity of introducing amino acid and properties of TvDAAO. Aromatic substitutions dramatically increased thermal stability of TvDAAO in contrast to small hydrophilic, hydrophobic and charged amino acids. For instance, TvDAAO Met104Phe had 11 times higher period of half inactivation at 56 0C in comparison to wild type. To figure out the nature of positive stabilization effect of introducing aromatic amino acids seven double mutants as a combination of Met104Phe, Tyr, Trp, Ser with spatially closed Phe258Ser and Phe54Ser were obtained. Properties of double mutants will be discussed. Finally Met104Phe mutation led to drastic stabilization of TvDAAO was combined with previously obtained mutants to give additional stabilization effects and desired catalytic properties.