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Transforming a Fructan:Fructan 6G-Fructosyltransferase from Perennial Ryegrass into a Sucrose:Sucrose 1-Fructosyltransferase^1,[C]

UMR INRA UCBN 950 EVA, Ecophysiologie Végétale, Agronomie et Nutritions NCS, Université de Caen, 14032 Caen cedex, France (B.L., J.L., M.-P.P.); Katholieke Universiteit Leuven, Laboratorium voor Moleculaire Plantenfysiologie, B–3001 Leuven, Belgium (L.S., W.L., K.L.R., R.V., W.V.d.E.); Department of Primary Industries, Victorian AgriBiosciences Centre, Bundoora, Victoria 3083, Australia (G.S.); and CNRS UMR 6037 IFRMP 23, UFR des Sciences, Université de Rouen, 76821 Mont Saint Aignan, France (H.M.)

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.

² Present address: Cornell University, 262 Plant Science Building, Tower Road, Ithaca, NY 14850.

³ Present address: Laboratoire de Nutrition Azotée des Plantes, Unité de Recherche 511, Département de Biologie Végétale, Centre de Recherche de Versailles-Grignon, INRA, RD 10, Route de Saint-Cyr, 78026 Versailles cedex, France.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Wim Van den Ende (wim.vandenende{at}bio.kuleuven.be).

^[C] Some figures in this article are displayed in color online but in black and white in the print edition.

www.plantphysiol.org/cgi/doi/10.1104/pp.108.125559

^* Corresponding author; e-mail wim.vandenende{at}bio.kuleuven.be.

Received July 1, 2008; accepted October 22, 2008; published October 24, 2008.

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