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Transforming wheat vacuolar invertase into a high affinity sucrose:sucrose 1‐fructosyltransferase
Author(s) -
Schroeven Lindsey,
Lammens Willem,
Van Laere André,
Van den Ende Wim
Publication year - 2008
Publication title -
new phytologist
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.742
H-Index - 244
eISSN - 1469-8137
pISSN - 0028-646X
DOI - 10.1111/j.1469-8137.2008.02603.x
Subject(s) - sucrose , fructan , invertase , sugar phosphates , biochemistry , fructose , mutant , chemistry , enzyme , sugar , gene
Summary• 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 ~ 43 m m ) 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.