title: Study of the Structure-Function-Stability Relationships in Yeast D-amino Acid Oxidase: Hydrophobization of Alpha-Helices.
abstract: Hydrophobization of alpha-helices is one of the general approaches used for improving the thermal stability of enzymes. A total of 11 serine residues located in alpha-helices have been found based on multiple alignments of the amino acid sequences of D-amino acid oxidases from different organisms and the analysis of the 3D-structure of D-amino acid oxidase from yeast Trigonopsis variabilis (TvDAAO, EC 1.4.3.3). As a result of further structural analysis, eight Ser residues in 67, 77, 78, 105, 270, 277, 335, and 336 positions have been selected to be substituted with Ala. S78A and S270A substitutions have resulted in dramatic destabilization of the enzyme. Mutant enzymes were inactivated during isolation from cells. Another six mutant TvDAAOs have been highly purified and their properties have been characterized. The amino acid substitutions S277A and S336A destabilized the protein globule. The thermal stabilities of TvDAAO S77A and TvDAAO S335A mutants were close to that of the wild-type enzyme, while S67A and S105A substitutions resulted in approximately 1.5- and 2.0-fold increases in the TvDAAO mutant thermal stability, respectively. Furthermore, the TvDAAO S105A mutant showed on average a 1.2- to 3.0-fold higher catalytic efficiency with D-Asn, D-Tyr, D-Phe, and D-Leu as compared to the wild-type enzyme.
authors.affiliation: Department of Chemistry, M.V. Lomonosov Moscow State University; Leninskie gory, 1/3, Moscow, 119991, Russia ; Innovations and High Technologies MSU Ltd, Tsymlyanskaya str., 16-96, Moscow, 109559, Russia.
authors.forename: I V
authors.initials: IV
authors.lastname: Golubev
authors.affiliation: Innovations and High Technologies MSU Ltd, Tsymlyanskaya str., 16-96, Moscow, 109559, Russia ; A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninskiy ave., 33/2, Moscow, 119071, Russia.
authors.forename: N V
authors.initials: NV
authors.lastname: Komarova
authors.affiliation: Department of Chemistry, M.V. Lomonosov Moscow State University; Leninskie gory, 1/3, Moscow, 119991, Russia ; Innovations and High Technologies MSU Ltd, Tsymlyanskaya str., 16-96, Moscow, 109559, Russia.
authors.forename: K V
authors.initials: KV
authors.lastname: Ryzhenkova
authors.affiliation: Department of Chemistry, M.V. Lomonosov Moscow State University; Leninskie gory, 1/3, Moscow, 119991, Russia ; Innovations and High Technologies MSU Ltd, Tsymlyanskaya str., 16-96, Moscow, 109559, Russia.
authors.forename: T A
authors.initials: TA
authors.lastname: Chubar
authors.affiliation: Innovations and High Technologies MSU Ltd, Tsymlyanskaya str., 16-96, Moscow, 109559, Russia ; A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninskiy ave., 33/2, Moscow, 119071, Russia.
authors.forename: S S
authors.initials: SS
authors.lastname: Savin
authors.affiliation: Department of Chemistry, M.V. Lomonosov Moscow State University; Leninskie gory, 1/3, Moscow, 119991, Russia ; Innovations and High Technologies MSU Ltd, Tsymlyanskaya str., 16-96, Moscow, 109559, Russia ; A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninskiy ave., 33/2, Moscow, 119071, Russia.
authors.forename: V I
authors.initials: VI
authors.lastname: Tishkov
keywords: D-amino acid oxidase from yeast Trigonopsis variabilis; hydrophobization of alpha-helices; protein engineering; site-directed mutagenesis; substrate specificity; thermal stability