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

Fructosyltransferase mutants specify a function for the β-fructosidase motif of the... 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 β(2-6) linkage or a terminal fructose via a β(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 β(2-1) linkages. Thus the region containing the sucrose-binding box is directing the fructan type synthesized. In invertases, the β-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 β-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 β(2-1) linkages. This is in agreement with the observed activities of the chimerical proteins and indicates that the β-fructosidase motif of the sucrose-binding box is specifying the fructan type synthesized. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant Molecular Biology Springer Journals

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

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Publisher
Kluwer Academic Publishers
Copyright
Copyright © 2004 by Kluwer Academic Publishers
Subject
Life Sciences; Biochemistry, general; Plant Sciences; Plant Pathology
ISSN
0167-4412
eISSN
1573-5028
D.O.I.
10.1007/s11103-004-0276-1
Publisher site
See Article on Publisher Site

Abstract

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 β(2-6) linkage or a terminal fructose via a β(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 β(2-1) linkages. Thus the region containing the sucrose-binding box is directing the fructan type synthesized. In invertases, the β-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 β-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 β(2-1) linkages. This is in agreement with the observed activities of the chimerical proteins and indicates that the β-fructosidase motif of the sucrose-binding box is specifying the fructan type synthesized.

Journal

Plant Molecular BiologySpringer Journals

Published: Dec 30, 2004

References

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