Creating S-type characteristics in the F-type enzyme fructan:fructan 1-fructosyltransferase of Triticum aestivum L.
Invertases cleave sucrose in glucose and fructose, using water as an acceptor. Fructosyltransferases catalyse the transfer of a fructosyl residue between sucrose and/or fructan molecules. Plant fructosyltransferases (FTs) evolved from vacuolar invertases by small mutational changes, leading to differences in substrate specificity. The S-type of enzymes (invertases, sucrose:sucrose 1-fructosyltransferases or 1-SSTs, and sucrose:fructan 6-fructosyltransferases or 6-SFTs) prefer sucrose as the donor substrate while F-type enzymes (fructan:fructan 1-fructosyltransferases or 1-FFTs and fructan:fructan 6(G)-fructosyltransferases or 6(G)-FFTs) preferentially use fructan as the donor substrate. Recently, a functional Asp/Arg or Asp/Lys couple in the Hypervariable Loop (HVL) was suggested to be essential to keep Asp in a favourable orientation for binding sucrose as the donor substrate in S-type enzymes. However, the F-type enzyme 1-FFT of Triticum aestivum (Ta1-FFT) also contains the Asp/Arg couple in the HVL, although it prefers fructan as the donor substrate. In this paper, mutagenesis studies on Ta1-FFT are presented. In Ta1-SST, Tyr282 (the Asp281 homologue) seems to be essential in creating a tight H-bond Network (HBN) in which the Arg-residue of the Asp/Arg couple is held in a fixed position. This tight HBN is disrupted in Ta1-FFT, leading to a more flexible Arg-residue and a dysfunctional Asp/Arg couple. A single D281Y mutation in Ta1-FFT restored the tight HBN and introduced typical S-type characteristics. Conclusively, in wheat FTs Asp281 (and its homologues) is involved in donor substrate specificity.
Schroeven
Lindsey
L
Faculteit Wetenschappen, Departement Biologie, KU Leuven, Heverlee, Belgium.
Lammens
Willem
W
Kawakami
Akira
A
Yoshida
Midori
M
Van Laere
André
A
Van den Ende
Wim
W
D000595:Amino Acid Sequence; D006602:Hexosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014908:Triticum / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins
D006602:Hexosyltransferases; D010940:Plant Proteins; C114852:1,2-beta-fructan 1(F)-fructosyltransferase; C021451:inulosucrase; D001426:Bacterial Proteins