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Transforming a Fructan:Fructan 6G-Fructosyltransferase from Perennial Ryegrass into a Sucrose:Sucrose 1-Fructosyltransferase
- Source :
- Plant Physiology. 149:327-339
- Publication Year :
- 2008
- Publisher :
- Oxford University Press (OUP), 2008.
-
Abstract
- Fructosyltransferases (FTs) synthesize fructans, fructose polymers accumulating in economically important cool-season grasses and cereals. FTs might be crucial for plant survival under stress conditions in species in which fructans represent the major form of reserve carbohydrate, such as perennial ryegrass (Lolium perenne). Two FT types can be distinguished: those using sucrose (S-type enzymes: sucrose: sucrose 1-fructosyltransferase [1-SST], sucrose: fructan 6-fructosyltransferase) and those using fructans (F-type enzymes: fructan: fructan 1-fructosyltransferase [1-FFT], fructan: fructan 6G-fructosyltransferase [6G-FFT]) as preferential donor substrate. Here, we report, to our knowledge for the first time, the transformation of an F-type enzyme (6G-FFT/1-FFT) into an S-type enzyme (1-SST) using perennial ryegrass 6G-FFT/1-FFT (Lp6G-FFT/1-FFT) and 1-SST (Lp1-SST) as model enzymes. This transformation was accomplished by mutating three amino acids (N340D, W343R, and S415N) in the vicinity of the active site of Lp6G-FFT/1-FFT. In addition, effects of each amino acid mutation alone or in combination have been studied. Our results strongly suggest that the amino acid at position 343 (tryptophan or arginine) can greatly determine the donor substrate characteristics by influencing the position of the amino acid at position 340. Moreover, the presence of arginine-343 negatively affects the formation of neofructan-type linkages. The results are compared with recent findings on donor substrate selectivity within the group of plant cell wall invertases and fructan exohydrolases. Taken together, these insights contribute to our knowledge of structure/function relationships within plant family 32 glycosyl hydrolases and open the way to the production of tailor-made fructans on a larger scale. ispartof: Plant physiology vol:149 issue:1 pages:327-339 ispartof: location:United States status: published
- Subjects :
- ray-diffraction structure
Sucrose
Physiology
Molecular Sequence Data
Plant Science
Biology
Substrate Specificity
molecular characterization
cell-wall invertase
chemistry.chemical_compound
Fructan
lolium-perenne
Lolium
Genetics
Glycoside hydrolase
Amino Acid Sequence
Cloning, Molecular
fructosyltransferase genes
Plant Proteins
chemistry.chemical_classification
Binding Sites
bacillus-subtilis levansucrase
Tryptophan
food and beverages
Focus Issue on the Grasses
Fructose
Carbohydrate
biology.organism_classification
key enzyme
Amino acid
Kinetics
Hexosyltransferases
chemistry
Biochemistry
Mutagenesis, Site-Directed
roots cichorium-intybus
transglycosylation activity
chicory roots
Sequence Alignment
Subjects
Details
- ISSN :
- 15322548
- Volume :
- 149
- Database :
- OpenAIRE
- Journal :
- Plant Physiology
- Accession number :
- edsair.doi.dedup.....05c5600d5bbb274e3875181897b4668b