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Site‐directed mutagenesis and protein 3D‐homology modelling suggest a catalytic mechanism for UDP‐glucose‐dependent betanidin 5‐ O ‐glucosyltransferase from Dorotheanthus bellidiformis
Author(s) -
Hans Judith,
Brandt Wolfgang,
Vogt Thomas
Publication year - 2004
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/j.1365-313x.2004.02133.x
Subject(s) - glucosyltransferase , glucosyltransferases , biochemistry , homology modeling , chemistry , stereochemistry , mutagenesis , amino acid , catalytic triad , binding site , active site , site directed mutagenesis , enzyme , biology , mutant , gene
Summary In livingstone daisy ( Dorotheanthus bellidiformis ), betanidin 5‐ O ‐glucosyltransferase (UGT73A5) is involved in the regiospecific glucosylation of betanidin and various flavonols. Based on sequence alignments several amino acid candidates which might be essential for catalysis were identified. The selected amino acids of the functionally expressed protein, suggested to be involved in substrate binding and turnover, were substituted via site‐directed mutagenesis. The substitution of two highly conserved amino acids, Glu378, located in the proposed UDP‐glucose binding site, and His22, located close to the N‐terminus, led to the complete loss of enzyme activity. A 3D model of this regiospecific betanidin and flavonoid glucosyltransferase was constructed and the active site modelled. This model was based on the crystallographic structure of a bacterial UDP‐glucose‐dependent glucosyltransferase from Amycolatopsis orientalis used as a template and the generated null mutations. To explain the observed inversion in the configuration of the bound sugar, semiempirical calculations favour an S N ‐1 reaction, as one plausible alternative to the generally proposed S N ‐2 mechanism discussed for plant natural product glucosyltransferases. The calculated structural data do not only explain the abstraction of a proton from the acceptor betanidin, but further imply that the reaction mechanism might also involve a catalytic triad, with similarities described for the serine protease family.