Site-directed mutagenesis of D-amino acid oxidase from yeast Trigonopsis variabilisстатья

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[1] Site-directed mutagenesis of d-amino acid oxidase from yeast trigonopsis variabilis / N. V. Cherskova, S. V. Khoronenkova, M. A. Panteleev, V. I. Tishkov // Journal of Biotechnology. — 2010. — Vol. 150, no. S1. — P. S442–S442. D-amino acid oxidase is a FAD-containing enzyme catalyzing D-amino acids oxidation to corresponding α-keto acids. Being widely occurred in nature, DAAO plays important role in metabolism of various organisms, including human, and is highly essential for practical use as well. Wild-type DAAO has some drawbacks which restrict use of the enzyme in practice. Rational design approach is widely applied to improve properties of the enzyme, such as thermal stability and catalytic properties. Earlier in this laboratory DAAO from yeast Trigonopsis variabilis (TvDAAO) was cloned in E.coli cells. The recombinant enzyme was obtained in active and soluble form. Site directed mutagenesis of Cys108 residue in TvDAAO resulted in mutant enzymes with changed substrate specificity profile. Two mutant TvDAAOs showed increased catalytic efficiency towards Cephalosporin C and one mutant had higher thermal stability compared to wt-TvDAAO. Two crystals of the mutant TvDAAO were obtained and the X-ray structure of the enzyme was determined at 2.8 and 1.8Å resolution. That provided us ability for rational design of TvDAAO properties. Analysis of the TvDAAO structure showed that there are two separate enters to the active site of the enzyme for D-amino acid and oxygen, respectively. It was found that Cys298 residue is placed in the smaller enter for oxygen and the sulfur atom is in SO3 form. This finding correlates with data of Schraeder et al. (Appl.Microbiol.Biotechnol. 1996) about essential role of Cys298 for enzyme activity. Cys298residue was substituted for Ala and mutant TvDAAO Cys108Ala was used as template. Double substitution, Cys108Ala/Cys298Ala, resulted in crucial loss of enzyme thermal stability. Additional substitution was introduced to improve thermal stability of the double mutant. New TvDAAO with three mutations had the same Tm value as wild-type enzyme (54 oC). This mutant and wild-type TvDAAO showed different kinetics of thermal inactivation. Activity loss of wild-type TvDAAO is described with sum of two exponential functions, while dependence of residual activity of mutant TvDAAO fits to one exponential function. Production of new mutant TvDAAO with improved properties is in progress. [ DOI ]

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