title: Fructan 1-exohydrolases. beta-(2,1)-trimmers during graminan biosynthesis in stems of wheat? Purification, characterization, mass mapping, and cloning of two fructan 1-exohydrolase isoforms.
abstract: Graminan-type fructans are temporarily stored in wheat (Triticum aestivum) stems. Two phases can be distinguished: a phase of fructan biosynthesis (green stems) followed by a breakdown phase (stems turning yellow). So far, no plant fructan exohydrolase enzymes have been cloned from a monocotyledonous species. Here, we report on the cloning, purification, and characterization of two fructan 1-exohydrolase cDNAs (1-FEH w1 and w2) from winter wheat stems. Similar to dicot plant 1-FEHs, they are derived from a special group within the cell wall-type invertases characterized by their low isoelectric points. The corresponding isoenzymes were purified to electrophoretic homogeneity, and their mass spectra were determined by quadrupole-time-of-flight mass spectrometry. Characterization of the purified enzymes revealed that inulin-type fructans [beta-(2,1)] are much better substrates than levan-type fructans [beta-(2,6)]. Although both enzymes are highly identical (98% identity), they showed different substrate specificity toward branched wheat stem fructans. Although 1-FEH activities were found to be considerably higher during the fructan breakdown phase, it was possible to purify substantial amounts of 1-FEH w2 from young, fructan biosynthesizing wheat stems, suggesting that this isoenzyme might play a role as a beta-(2,1)-trimmer throughout the period of active graminan biosynthesis. In this way, the species and developmental stage-specific complex fructan patterns found in monocots might be determined by the relative proportions and specificities of both fructan biosynthetic and breakdown enzymes.
authors.affiliation: Department of Biology, Laboratory of Neuro-endocrinology and Immunological Biotechnology, Zoological Institute, KULeuven, Naamsestraat 59, B-3000 Leuven, Belgium. wim.vandenende@bio.kuleuven.ac.be
authors.forename: Wim
authors.initials: W
authors.lastname: Van Den Ende
authors.forename: Stefan
authors.initials: S
authors.lastname: Clerens
authors.forename: Rudy
authors.initials: R
authors.lastname: Vergauwen
authors.forename: Liesbet
authors.initials: L
authors.lastname: Van Riet
authors.forename: André
authors.initials: A
authors.lastname: Van Laere
authors.forename: Midori
authors.initials: M
authors.lastname: Yoshida
authors.forename: Akira
authors.initials: A
authors.lastname: Kawakami
mesh_terms: D000595:Amino Acid Sequence; D002473:Cell Wall / Q000201:enzymology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D006026:Glycoside Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010802:Phylogeny; D018547:Plant Stems / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014908:Triticum / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D043324:beta-Fructofuranosidase
chemical_list: D018076:DNA, Complementary; D007527:Isoenzymes; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase; C044118:fructan beta-fructosidase