Using Natural Variation to Investigate the Function of Individual Amino Acids in the Sucrose-Binding Box of Fructan:Fructan 6G-Fructosyltransferase (6G-FFT) in Product Formation

Abstract

Enzymes of the glycosyl hydrolase family 32 are highly similar with respect to primary sequence but catalyze divergent reactions. Previously, the importance of the conserved sucrose-binding box in determining product specificity of onion fructan:fructan 6G-fructosyltransferase (6G-FFT) was established [Ritsema et al., 2004, Plant Mol. Biol. 54: 853-863]. Onion 6G-FFT synthesizes the complex fructan neo-series inulin by transferring fructose residues to either a terminal fructose or a terminal glucose residue. In the present study we have elucidated the molecular determinants of product specificity by substitution of individual amino acids of the sucrose binding box with amino acids that are present on homologous positions in other fructosyltransferases or vacuolar invertases. Substituting the presumed nucleophile Asp85 of the beta-fructosidase motif resulted in an inactive enzyme. 6G-FFT mutants S87N and S87D did not change substrate or product specificities, whereas mutants N84Y and N84G resulted in an inactive enzyme. Most interestingly, mutants N84S, N84A, and N84Q added fructose residues preferably to a terminal fructose and hardly to the terminal glucose. This resulted in the preferential production of inulin-type fructans. Combining mutations showed that amino acid 84 determines product specificity of 6G-FFT irrespective of the amino acid at position 87.

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

“…Replacement of Asn-84 (structurally equivalent to Gly59 in AjFT) with Ser, Ala, or Gln turns onion 6G-FFT into 1-SST, indicating that Asn-84 determines the product specificity (48). Mutants W161Y and N166S of onion invertase in this region showed enhanced transfructosylation activities (49).…”

“…However, Asn-252, located at blade II of B. megaterium levansucrase, and Arg-433 and Tyr-429, located at blade V of B. subtilis levansucrase, are related to the synthesis of polymer versus oligosaccharide (48,49). According to the raffinose binding in AjFT, the polar groups at the rim of the active-site pocket near the contact between blades II and III might be the critical location for seeking particular residues responsible for activity of this kind.…”

Molecular Dissection of Variation in Carbohydrate Metabolism Related to Water-Soluble Carbohydrate Accumulation in Stems of Wheat

“…In this sense chloroplast Fru-bisP genes are excluded from the cytoplasmic Fru-bisP family, as the former is involved in the Calvin cycle and the latter is involved in gluconeogenesis. Extensive reannotation is given for genes from glycosyltransferase family 32, which contains cell wall and vacuolar invertases, fructosyltransferases, and fructan exohydrolases (FEHs), based on recent publications (Chalmers et al, 2003(Chalmers et al, , 2005Van den Ende et al, 2003De Coninck et al, 2005;Ritsema et al, 2005Ritsema et al, , 2006Francki et al, 2006;Lasseur et al, 2006;Van Riet et al, 2006;Ji et al, 2007;Le Roy et al, 2007;Lothier et al, 2007;Verhaest et al, 2007 To examine the relationship between the transcript levels of individual carbohydrate metabolic genes and stem WSC levels, 16 Affymetrix wheat GeneChips were used to determine the transcript levels of carbohydrate metabolic genes in the stem of eight SB lines at anthesis with two field replicates of each line. Correlation analysis between the transcript levels of individual genes and stem WSC concentrations was performed using both genotypic means (n 5 8) and individual samples (8 genotypes 3 2 field replicates, n 5 16; Supplemental Table S1).…”

Food Biochemical Study on Fructans and Related Synthesis Enzymes

“…These results suggest that amino acid substitution in the β fructosidase motif of wild type AoFT1 altered substrate specificity, from 6G FFT to 1 FFT, and was similar to those observed in onion bulbs. 35) 3. Post-harvest metabolism of fructo-oligosaccharides in onion and asparagus plants Alongside the studies on fructo oligodsaccharide accumulation during seed development 36) and bulbing, 37) we also studied the main enzyme activities involved in the synthesis and the hydrolysis of fructo oligosaccharides during the pre harvest 38) (growth), and post harvest 38,39) (storage) life of onion bulbs and asparagus spears, 40) although present knowledge is insufficient to clearly explain the mechanisms triggering enzymes or the mechanisms by which fructo oligosaccharides contribute to bulb quality and perishability.…”