ИСТИНА

D-amino acid oxidase is a FAD-containing enzyme catalyzing D-amino acids oxidation to corresponding α-keto acids. DAAO plays important role in regulation of many proc-esses in living cell (especially in mammals). The enzyme is also of high practical inter-est for pharmaceutical industry, fine organic synthesis and analytical biotechnology. It is used for biocatalytic conversion of cephalosporin C into 7-amino cephalosporanic acid (7-ACA) being the key precursor for synthesis of cephalosporanic antibiotics. D-amino acids oxidase from the yeast Trigonopsis variabilis (TvDAAO) is the most ap-propriate enzyme for application due to the best properties among all known DAAO’s. Nevertheless wild type TvDAAO often possesses drawbacks for practical processes. Properties of enzyme can be improved by protein design technique. Here we will present the results of protein engineering of TvDAAO. Computer analysis of TvDAAO structure and docking of substrates to active site revealed flexible amino acid residues, including Met104, located at the entrance and controlling access of substrates to the active site. To clear out the role of Met104 residue in catalysis and thermal stability eight substitutions to small and bulky amino acids were proposed based on computer simulation of potential mutants. Site-directed mutagenesis of Met104 was performed to introduce chosen substitutions. All mutants were obtained in soluble and active form. Study of properties showed that volume of residue in the 104th position has a crucial influence on thermal stability and catalytic activity. So introduc-tion of bulky residues led to significant changes of thermal stability and affected on catalytic efficiency with the most of substrates. To investigate interaction between Met104 and spatially closed Phe54 several double mutants were obtained and character-ized. The work is supported by Russian Foundation for Basic Research (grant #11-04-00959-а) and grant “U.M.N.I.K.” (I.V.Golubev) from the Russian Foundation for Assis-tance to Small Innovative Enterprises (FASIE).