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首页> 外文期刊>Biochemistry >Engineering of Substrate Specificity of D-Amino Acid Oxidase from the Yeast Trigonopsis variabilis: Directed Mutagenesis of Phe258 Residue
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Engineering of Substrate Specificity of D-Amino Acid Oxidase from the Yeast Trigonopsis variabilis: Directed Mutagenesis of Phe258 Residue

机译：酵母Trigonopsis variabilis的D-氨基酸氧化酶的底物特异性工程：Phe258残留物的定向诱变

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摘要

Natural D-amino acid oxidases (DAAO) are not suitable for selective determination of D-amino acids due to their broad substrate specificity profiles. Analysis of the 3D-structure of the DAAO enzyme from the yeast Trigonopsis variabilis (TvDAAO) revealed the Phe258 residue located at the surface of the protein globule to be in the entrance to the active site. The Phe258 residue was mutated to Ala, Ser, and Tyr residues. The mutant TvDAAOs with amino acid substitutions Phe258Ala, Phe258Ser, and Phe258Tyr were purified to homogeneity and their thermal stability and substrate 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 substitutions caused dramatic changes in substrate specificity. Increasing the side chain size with the Phe258Tyr substitution decreased the kinetic parameters with all the D-amino acids studied. For the two other substitutions, 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 efficiency 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.
机译：天然D-氨基酸氧化酶（DAAO）由于其广泛的底物特异性特征而不适用于D-氨基酸的选择性测定。对酵母Trigonopsis variabilis（TvDAAO）的DAAO酶的3D结构进行分析后发现，位于蛋白质小球表面的Phe258残基位于活性位点的入口。 Phe258残基突变为Ala，Ser和Tyr残基。将具有氨基酸取代的Phe258Ala，Phe258Ser和Phe258Tyr的突变TvDAAOs纯化至均一，并研究其热稳定性和底物特异性。这些取代导致酶的轻微稳定化（Phe258Tyr）或去稳定化（Phe258Ser）。半灭活期的变化小于两倍。但是，这些取代引起底物特异性的显着变化。用Phe258Tyr取代增加侧链大小会降低所有研究的D-氨基酸的动力学参数。对于另外两个替代，底物特异性谱变窄。催化效率仅对于D-Tyr，D-Phe和D-Leu有所提高，而对于所有其他D-氨基酸，该参数急剧降低。使用D-Tyr，D-Phe和D-Leu对TvDAAO Phe258Ala的催化效率提高了3.66、11.7-和1.5倍，对于TvDAAO Phe258Ser则提高了1.7-，4.75-和6.61-分别。

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