title: Chain Elongation of raffinose in pea seeds. Isolation, characterization, and molecular cloning of mutifunctional enzyme catalyzing the synthesis of stachyose and verbascose.
abstract: Raffinose oligosaccharides are major soluble carbohydrates in seeds and other tissues of plants. Their biosynthesis proceeds by stepwise addition of galactose units to sucrose, which are provided by the unusual donor galactinol (O-alpha-d-galactopyranosyl-(1-->1)-l-myo-inositol). Chain elongation may also proceed by transfer of galactose units between raffinose oligosaccharides. We here report on the purification, characterization, and heterologous expression of a multifunctional stachyose synthase (EC ) from developing pea (Pisum sativum L.) seeds. The protein, a member of family 36 of glycoside hydrolases, catalyzes the synthesis of stachyose, the tetrasaccharide of the raffinose series, by galactosyl transfer from galactinol to raffinose. It also mediates the synthesis of the pentasaccharide verbascose by galactosyl transfer from galactinol to stachyose as well as by self-transfer of the terminal galactose residue from one stachyose molecule to another. These activities show optima at pH 7.0. The enzyme also catalyzes hydrolysis of the terminal galactose residue of its substrates, but is unable to initiate the synthesis of raffinose oligosaccharides by galactosyl transfer from galactinol to sucrose. A minimum reaction mechanism which accounts for the broad substrate specificity and the steady-state kinetic properties of the protein is presented.
authors.affiliation: Institute of Ecology, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria.
authors.forename: Thomas
authors.initials: T
authors.lastname: Peterbauer
authors.forename: Jan
authors.initials: J
authors.lastname: Mucha
authors.forename: Lukas
authors.initials: L
authors.lastname: Mach
authors.forename: Andreas
authors.initials: A
authors.lastname: Richter
mesh_terms: D000595:Amino Acid Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D005700:Galactosyltransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007294:Inositol / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000096:biosynthesis*; D018532:Pisum sativum / Q000378:metabolism*; D011887:Raffinose / Q000096:biosynthesis*; D013379:Substrate Specificity
chemical_list: D009844:Oligosaccharides; C005695:stachyose; D007294:Inositol; D005700:Galactosyltransferases; C113913:galactinol -raffinose galactosyltransferase; D011887:Raffinose