title: Transforming wheat vacuolar invertase into a high affinity sucrose:sucrose 1-fructosyltransferase.
abstract: Vacuolar invertases (VIs) degrade sucrose to glucose and fructose. Additionally, the fructan plant wheat (Triticum aestivum) contains different fructosyltransferases (FTs), which have evolved from VIs by developing the capacity to bind sucrose or fructans as acceptor substrates. Modelling studies revealed a hydrogen bonding network in the conserved WMNDPNG motif of VIs, which is absent in FTs. In this study, the hydrogen bonding network of wheat VI was disrupted by site-directed mutagenesis in the 23WMNDPNG29 motif. While the single mutants (W23Y, N25S) showed a moderate increase in 1-kestose production, a synergistic effect was observed for the double mutant (W23Y+N25S), showing a 17-fold increase in transfructosylation capacity, and becoming a real sucrose:sucrose 1-fructosyltransferase. Vacuolar invertases are fully saturable enzymes, contrary to FTs. This is the first report on the development of a fully saturable FT with respect to 1-kestose formation. The superior kinetics (K(m) approximately 43 mM) make the enzyme useful for biotechnological applications. The results indicate that changes in the WMNDPNG motif are necessary to develop transfructosylating capability. The shift towards smaller and/or more hydrophilic residues in this motif might contribute to the formation of a specific acceptor site for binding of sugar, instead of water.
authors: K. U. Leuven, Laboratorium voor Moleculaire Plantenfysiologie, Kasteelpark Arenberg 31, bus 2434, B-3001 Leuven, Belgium.
authors: Lindsey
authors: L
authors: Schroeven
authors: Willem
authors: W
authors: Lammens
authors: André
authors: A
authors: Van Laere
authors: Wim
authors: W
authors: Van den Ende
mesh_terms: D000595:Amino Acid Sequence; D019143:Evolution, Molecular; D005630:Fructans / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D006602:Hexosyltransferases / Q000096:biosynthesis* / Q000737:chemistry; D006859:Hydrogen / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014908:Triticum / Q000201:enzymology* / Q000235:genetics; D014617:Vacuoles / Q000378:metabolism; D043324:beta-Fructofuranosidase / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins
chemical_list: D005630:Fructans; D006602:Hexosyltransferases; D006859:Hydrogen; D043324:beta-Fructofuranosidase; C021451:inulosucrase; D001426:Bacterial Proteins