TAILIEUCHUNG - Báo cáo khoa học: Probing suggested catalytic domains of glycosyltransferases by site-directed mutagenesis Tobias Hefner and Joachim Stockigt ¨

The plant enzyme arbutin synthase isolated from cell sus-pension cultures ofRauvolfia serpentinaand heterologously expressed inEscherichia coliis a member of the NRD1b family of glycosyltransferases. This enzyme was used to prove, by site-directed mutagenesis, suggested catalytic domains and reaction mechanisms proposed for enzyme-catalyzed glycosylation. Replacement of amino acids far from the NRD domain do not significantly affect arbutin synthase activity. | Eur. J. Biochem. 270 533-538 2003 FEBS 2003 doi Probing suggested catalytic domains of glycosyltransferases by site-directed mutagenesis Tobias Hefner and Joachim Stockigt Lehrstuhlfur Pharmazeutische Biologie Johannes Gutenberg-Universităt Mainz Germany The plant enzyme arbutin synthase isolated from cell suspension cultures of Rauvolfia serpentina and heterologously expressed in Escherichia coli is a member of the NRD1b family of glycosyltransferases. This enzyme was used to prove by site-directed mutagenesis suggested catalytic domains and reaction mechanisms proposed for enzyme-catalyzed glycosylation. Replacement of amino acids far from the NRD domain do not significantly affect arbutin synthase activity. Exchange of amino acids at the NRD site leads to a decrease of enzymatic activity . substitution of Glu368 by Asp. Glu368 which is a conserved amino acid in glycosyltransferases located at position 2 and is important for enzyme activity does not serve as the nucleophile in the catalytic centre as proposed. When it is replaced by Ala the resulting mutant enzyme E368A exhibits comparable activity as found for E368D in respect to vanillin. Enzyme activities of wild-type and E368A towards several substrates were not affected at the same level. His360 at position 1 of NRD1 b glycosyltransferases occupies a more crucial role as expected. When it is exchanged against other basic amino acids such as Lys or Arg the enzyme activity decreases 1000-fold. Replacement of His360 by Glu leads to a mutant enzyme H360E with an w 4000-fold lower activity compared with the wild-type. This mutein still produces a b-glucoside not an a-glucoside and therefore indicates that generation of the typical E-E motif of NRD1a glycosyltransferases does not convert a NRD1b enzyme into a NRD1a enzyme. The presented data do not support several suggestions made in the literature about catalytic amino acids involved in the glycosyltransfer reaction. Keywords .

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