Transforming a Fructan:Fructan 6G-Fructosyltransferase from Perennial Ryegrass into a Sucrose:Sucrose 1-Fructosyltransferase  

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 , sucrose:fructan 6-fructosyltransferase) and those using fructans (F-type enzymes: fructan:fructan 1-fructosyltransferase , 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.Fructans are Fru polymers that can be considered as an extension of Suc, accumulating above a certain threshold Suc concentration (Cairns and Pollock, 1988;Guerrand et al., 1996;Maleux and Van den Ende, 2007). Fructans occur in many dicot and monocot plant species mainly belonging to Asteraceae, Liliaceae, and Poaceae (Hendry, 1993;Prud'homme et al., 2007;Shiomi et al., 2007;Van den Ende and Van Laere, 2007;Yoshida et al., 2007). Nowadays, research is mainly focused on economically important cereals (wheat [Triticum aestivum] and barley [Hordeum vulgare]) and forage grasses (mainly Lolium species). Depending on the linkage type between the fructosyl residues and the position of the Glc residue (Lewis, 1993), several fructan types can be distinguished. Fructans with a terminal Glc residue include the b(2-1)-type fructans (inulin, principally occurring in dicots) and the linear b(2-6) (levan)-or branched-type fructans (graminan) with both b(2-6) and b(2-1) linkages (as occurring in bacteria and cereals, respectively). Fructans with an internal Glc residue include the neoinulin and neolevan types (occurring in monocots such as Allium, Asparagus, and Lolium).Apart from their function as a vacuolar storage carbohydrate, fructans may help to protect plants ag...

Development of rubber‐enriched dandelion varieties by metabolic engineering of the inulin pathway

“…Multiple sequence alignments using MUSCLE revealed the presence of three GH32 family‐specific conserved regions including the three catalytically active amino acids shown in bold: x‐x‐x‐ D ‐P‐D/N‐G; R D P; and E C (Altenbach and Ritsema, 2007; Altenbach et al ., 2005; Edgar, 2004; Schroeven et al ., 2008) (Figure S1). Furthermore, the fructosyltransferase‐specific motif x‐A/G‐Y/F was found in Tb1‐SST, Tk1‐SST, Tb1‐FFT and Tk1‐FFT (Altenbach et al ., 2005; Lasseur et al ., 2009). …”

“…Both dandelion 1‐FEHs showed lower levels of identity with the chicory enzymes Ci1‐FEHI (CAC19366) with 52% identity, and an invertase (CAA72009) with 59% identity. The hydrolase‐specific W‐A/S/G‐W motif and the three conserved regions common to GH32 enzymes, including the three highly active amino acids mentioned above, were also found in Tb1‐FEH and Tk1‐FEH (Altenbach et al ., 2005; Lasseur et al ., 2009; Le Roy et al ., 2007, 2008) (Figure S1).…”

Crystal Structures of Aspergillus japonicus Fructosyltransferase Complex with Donor/Acceptor Substrates Reveal Complete Subsites in the Active Site for Catalysis

“…Moreover, substitutions of Asn-340/Trp-343 of perennial ryegrass 6G-fructosyltransferase at the positions to Asp/Arg transformed the enzyme into 1-SST (Fig. 2) (55). The Asp/Arg(Lys) pair located at the loop connecting the second and third strands of blade IV (corresponding to Glu-318/His-332 in AjFT) and the hydrogen bond network created by this D/R pair were thus suggested for recognition of sucrose as a substrate (53)(54)(55).…”

“…The mutagenesis in search of the important residues responsible for substrate recognition and the type of catalytic reaction has been studied in plant GH32 members and GH68 microbial levansucrases (51)(52)(53)(54)(55)(56)(57)(58). The superimposed structures show that Glu-318 in AjFT is at a position equivalent to Asp-239 in AtcwINV, which has been shown to be critical for binding and stabilizing sucrose (Fig.…”

“…2) (55). The Asp/Arg(Lys) pair located at the loop connecting the second and third strands of blade IV (corresponding to Glu-318/His-332 in AjFT) and the hydrogen bond network created by this D/R pair were thus suggested for recognition of sucrose as a substrate (53)(54)(55). The equivalent residue at this position is Phe-233 in fructan 6-exohydrolase of the sugar beet Beta vulgaris.…”