title: Indole-3-acetic acid UDP-glucosyltransferase from immature seeds of pea is involved in modification of glycoproteins.
abstract: The glycosylation of auxin is one of mechanisms contributing to hormonal homeostasis. The enzyme UDPG: indole-3-ylacetyl-β-D-glucosyltransferase (IAA glucosyltransferase, IAGlc synthase) catalyzes the reversible reaction: IAA+UDPG↔1-O-IA-glucose+UDP, which is the first step in the biosynthesis of IAA-ester conjugates in monocotyledonous plants. In this study, we report IAA-glucosyltransferase isolated using a biochemical approach from immature seed of pea (Pisum sativum). The enzyme was purified by PEG fractionation, DEAE-Sephacel anion-exchange chromatography and preparative PAGE. LC-MS/MS analysis of tryptic peptides of the enzyme revealed the high identity with maize IAGlc synthase, but lack of homology with other IAA-glucosyltransferases from dicots. Biochemical characterization showed that of several acyl acceptors tested, the enzyme had the highest activity on IAA as the glucosyl acceptor (Km=0.52 mM, Vmax=161 nmol min(-1), kcat/Km=4.36 mM s(-1)) and lower activity on indole-3-propionic acid and 1-naphthalene acetic acid. Whereas indole-3-butyric acid and indole-3-propionic acid were competitive inhibitors of IAGlc synthase, D-gluconic acid lactone, an inhibitor of β-glucosidase activity, potentiated the enzyme activity at the optimal concentration of 0.3mM. Moreover, we demonstrated that the 1-O-IA-glucose synthesized by IAGlc synthase is the substrate for IAA labeling of glycoproteins from pea seeds indicating a possible role of this enzyme in the covalent modification of a class of proteins by a plant hormone.
authors.affiliation: Department of Biochemistry, Nicolaus Copernicus University, Lwowska 1, 87-100 Torun, Poland. Electronic address: maciejost@umk.pl.
authors.forename: Maciej
authors.initials: M
authors.lastname: Ostrowski
authors.affiliation: Department of Biochemistry, Nicolaus Copernicus University, Lwowska 1, 87-100 Torun, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Hetmann
authors.affiliation: Department of Biochemistry, Nicolaus Copernicus University, Lwowska 1, 87-100 Torun, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Jakubowska
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel; D005942:Gluconates; D005964:Glucosyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D006004:Glycogen Debranching Enzyme System; D006023:Glycoproteins / Q000032:analysis / Q000187:drug effects / Q000378:metabolism*; D006031:Glycosylation; D007210:Indoleacetic Acids / Q000378:metabolism*; D007211:Indoles; D018532:Pisum sativum / Q000737:chemistry* / Q000196:embryology / Q000235:genetics; D010937:Plant Growth Regulators / Q000378:metabolism; D012639:Seeds / Q000737:chemistry* / Q000201:enzymology; D045868:Sorghum / Q000235:genetics / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D014532:Uridine Diphosphate Glucose / Q000378:metabolism; D003313:Zea mays / Q000235:genetics / Q000378:metabolism
chemical_list: D005942:Gluconates; D006004:Glycogen Debranching Enzyme System; D006023:Glycoproteins; D007210:Indoleacetic Acids; D007211:Indoles; D010937:Plant Growth Regulators; C014612:indolebutyric acid; C030737:indoleacetic acid; C030374:indole; D005964:Glucosyltransferases; C071643:indole-3-acetate beta-glucosyltransferase; C020569:4 alpha-glucanotransferase; C030691:gluconic acid; D014532:Uridine Diphosphate Glucose
keywords: Auxin conjugate; Glucosylation; Glucosyltransferase; Indole-3-acetic acid; Indole-3-ylacetyl-β-d-glucose; Pisum sativum