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.
Ritsema
Tita
T
Department of Molecular Plant Physiology, University Utrecht, Padualaan 8, CH, The Netherlands. Tita.Ritsema@unibas.ch
Verhaar
Auke
A
Vijin
Irma
I
Smeekens
Sjef
S
D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D005630:Fructans / Q000096:biosynthesis*; D006602:Hexosyltransferases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013395:Sucrose / Q000378:metabolism*; D014312:Trisaccharides / Q000378:metabolism; D043324:beta-Fructofuranosidase / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins
D005630:Fructans; D006602:Hexosyltransferases; D013395:Sucrose; D014312:Trisaccharides; D043324:beta-Fructofuranosidase; C072433:1-kestose; C021451:inulosucrase; D001426:Bacterial Proteins