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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
Abstract
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 1.4.3.3) 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.
Copyright (C) Chemistry Dept., Moscow State University, 2002
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