Probing suggested catalytic domains of glycosyltransferases by site-directed mutagenesis.
The plant enzyme arbutin synthase isolated from cell suspension cultures of Rauvolfia serpentina and heterologously expressed in Escherichia coli is a member of the NRD1beta 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, e.g. 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 NRD1beta 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 approximately 1000-fold. Replacement of His360 by Glu leads to a mutant enzyme (H360E) with an approximately 4000-fold lower activity compared with the wild-type. This mutein still produces a beta-glucoside, not an alpha-glucoside and therefore indicates that generation of the typical E-E motif of NRD1alpha glycosyltransferases does not convert a NRD1beta enzyme into a NRD1alpha enzyme. The presented data do not support several suggestions made in the literature about catalytic amino acids involved in the glycosyltransfer reaction.
Hefner
Tobias
T
Lehrstuhl für Pharmazeutische Biologie, Johannes Gutenberg-Universität Mainz, Germany.
Stöckigt
Joachim
J
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001104:Arbutin / Q000378:metabolism; D001665:Binding Sites; D020134:Catalytic Domain; D017124:Conserved Sequence; D017931:DNA Primers / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic; D016695:Glycosyltransferases / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011926:Rauwolfia / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D017931:DNA Primers; D011994:Recombinant Proteins; D001104:Arbutin; D016695:Glycosyltransferases