• 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 sitedirected
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
• 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 (Km ~ 43 mM) make the enzyme useful for biotechnological
• 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.