Fructosyltransferase mutants specify a function for the beta-fructosidase motif of the sucrose-binding box in specifying the fructan type synthesized.

The onion fructosyltransferase fructan:fructan 6G-fructosyltransferase (6G-FFT) synthesizes fructans of the inulin neo-series using 1-kestose as a substrate. 6G-FFT couples a fructosyl residue to either the terminal glucose via a beta (2-6) linkage or a terminal fructose via a beta (2-1) linkage. The sucrose-binding box is present at the N-terminus of invertases and fructosyltransferases. We tested its function by producing swaps of the first 36 amino acids of 6G-FFT with that of onion sucrose:sucrose 1-fructosyltransferase (1-SST) (SST-GFT) and vacuolar invertase (INV-GFT). In contrast to 6G-FFT, invertase and 1-SST are able to utilize sucrose as their only substrate. The chimerical enzymes were unable to use sucrose, but were active when incubated with 1-kestose. INV-GFT synthesized a similar array of fructans as 6G-FFT, in contrast, SST-GFT showed a dramatic shift in activity towards synthesis of beta (2-1) linkages. Thus the region containing the sucrose-binding box is directing the fructan type synthesized. In invertases, the beta -fructosidase motif, which is part of the sucrose-binding box, consists of NDPNG/A. This motif is variable in fructosyltransferases and consists of NDPSG in 6G-FFT and ADPNA in 1-SST of onion. We studied the importance of the 6G-FFT beta -fructosidase motif using mutants S87N (NDPNG) and N84A;S87N (ADPNG). S87N has 6G-FFT activity, whereas N84A;S87N has a activity that was shifted towards synthesis of beta (2-1) linkages. This is in agreement with the observed activities of the chimerical proteins and indicates that the beta -fructosidase motif of the sucrose-binding box is specifying the fructan type synthesized.