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Интеллектуальная Система Тематического Исследования НАукометрических данных |
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Catalytic properties of mutant D-amino acid oxidase with three amino acid substitutionsтезисы доклада
- Авторы: Karnaukhov V.K., Zarubina S.A., Golubev I.V., Osipova T.A., Savin S.S., Tishkov V.I.
- Сборник: Program and Abstract Book of International Conference "Biocatalysis-2015: Fundamentals and Applications", Moscow region, Avantel Club Istra
- Тезисы
- Год издания: 2015
- Место издания: Innovations and High Technologies MSU Ltd Moscow
- Первая страница: 109
- Последняя страница: 109
- Аннотация: D-Amino acid oxidase (DAAO) is a FAD-containing enzyme that oxidizes D-amino acids to corresponding imino acids, followed by hydrolysis of imino acid to α–keto acid ammonium ion. The en-zyme plays different important physiological functions in living organisms. Besides, the enzyme has various practical applications in biotechnology and medical diagnostics. Gene of DAAO from the yeast Trigonopsis variabilis (TvDAAO) was cloned and expressed in E. coli. Rational design approach was used to study the structure-function relationship and to improve catalytic properties and temperature stability of TvDAAO. 3D structure of mutant enzyme was deter-mined with resolution 1.8Å and it was used for selection of amino acid changes. Different single-point mutants of TvDAAO were prepared and characterized. Positive amino acid substitutions (two muta-tions in coenzyme biding domain plus different single changes in substrate binding domain) were com-bined. In this work catalytic parameters (kcat, KM, kcat/KM) of the mutant enzyme with three amino acid changes (TvDAAO 3M) were determined. It was shown that catalytic properties of substrates with bulky side chain (D-Phe, D-Tyr, D-Trp) considerably worsened compared to wild-type enzyme. It can be caused by fact that the substitution of methionine to phenylalanine residue in active site results in steric hindrance for substrates in the entrance to the active site. The parameter of catalytic efficiency with D-Ser increased about 3 times compared to wild-type enzyme. We have shown that the profile of substrate specificity of triple mutant is similar to the profile of the mutant with the substitution in the active site. In was found that the substitution in the cofactor-binding domain results in further improvement of catalytic parameters with some substrates. The work is carried out with the support of the Russian Foundation for Basic Research (grants 14-04-00865-a and 14-04-31194-mol_а).
- Добавил в систему: Тишков Владимир Иванович