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D. L. Atroshenko, I. V. Golubev, S. S. Savin, V. I. Tishkov

Influence of Met/Leu amino acid changes on catalytic properties and oxidative and thermal stability of yeast D-amino acid oxidase


Oxidative stability of enzymes is mostly dependent on stability of Cys and Met residues. Three single point mutants with changes Met/Leu of D-amino acid oxidase (DAAO, EC from the yeast Trigonopsis variabilis (TvDAAO) were prepared and characterized. Selection of position for amino acid residue substitution was made based on multiple alignment of different DAAO amino acid sequences and analysis of the TvDAAO structure. It was shown that substrate specificity profile changed for all mutans. KM values for small and bulky D-amino acids increased and decreased, respectively. In one case change Met/Leu resulted in 2-3-fold increase of thermal stability. Method to determine stability of TvDAAO in presence of hydrogen peroxide was developed and oxidative stability of wild-type and mutant TvDAAOs was studied. It was shown that all three mutations did not change of the enzyme oxidative stability.
Key words: D-amino acid oxidase, Trigonopsis variabilis, site-directed mutagenesis, oxidative stability, thermal stability, catalytic properties.
Moscow University Chemistry Bulletin.
2016, Vol. 57, No. 4, P. 253

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