ENGINEERING OF SUBSTRATE SPECIFICITY OF D-AMINO ACID OXIDASE FROM YEAST <i>Trigonopsis variabilis</i>: DIRECTED MUTAGENESIS OF Phe258 RESIDUE
Natural D-amino acid oxidases (DAAO) are not suitable for selective determination of D-amino acids due to their wide substrate specificity profiles. Analysis of the 3D-structure of the DAAO enzyme from the yeast Trigonopsis vari- abilis (TvDAAO) revealed the Phe258 residue located at the surface of the protein globule in the entrance to the active site. The Phe258 residue was changed to Ala, Ser, and Tyr residues. The mutant TvDAAOs with amino acid substitu- tions Phe258Ala, Phe258Ser, and Phe258Tyr were purified to homogeneous form and their thermal stability and sub- strate specificity were studied. These substitutions resulted in either slight stabilization (Phe258Tyr) or destabilization (Phe258Ser) of the enzyme. The change in half-inactivation periods was less than twofold. However, these substitu- tions caused dramatic changes in the substrate specificity profiles. Enlargement of side chain size due to the Phe258Tyr substitution decreased the kinetic parameters with all the D-amino acids studied. For the two other sub- stitutions, the substrate specificity profiles narrowed. The catalytic efficiency increased only for D-Tyr, D-Phe, and D-Leu, and for all other D-amino acids this parameter dramatically decreased. The improvement of catalytic effi- ciency with D-Tyr, D-Phe, and D-Leu for TvDAAO Phe258Ala was 3.66-, 11.7-, and 1.5-fold, and for TvDAAO Phe258Ser it was 1.7-, 4.75-, and 6.61-fold, respectively.