title: Characterization and expression profile of two UDP-glucosyltransferases, UGT85K4 and UGT85K5, catalyzing the last step in cyanogenic glucoside biosynthesis in cassava.
abstract: Manihot esculenta (cassava) contains two cyanogenic glucosides, linamarin and lotaustralin, biosynthesized from l-valine and l-isoleucine, respectively. In this study, cDNAs encoding two uridine diphosphate glycosyltransferase (UGT) paralogs, assigned the names UGT85K4 and UGT85K5, have been isolated from cassava. The paralogs display 96% amino acid identity, and belong to a family containing cyanogenic glucoside-specific UGTs from Sorghum bicolor and Prunus dulcis. Recombinant UGT85K4 and UGT85K5 produced in Escherichia coli were able to glucosylate acetone cyanohydrin and 2-hydroxy-2-methylbutyronitrile, forming linamarin and lotaustralin. UGT85K4 and UGT85K5 show broad in vitro substrate specificity, as documented by their ability to glucosylate other hydroxynitriles, some flavonoids and simple alcohols. Immunolocalization studies indicated that UGT85K4 and UGT85K5 co-occur with CYP79D1/D2 and CYP71E7 paralogs, which catalyze earlier steps in cyanogenic glucoside synthesis in cassava. These enzymes are all found in mesophyll and xylem parenchyma cells in the first unfolded cassava leaf. In situ PCR showed that UGT85K4 and UGT85K5 are co-expressed with CYP79D1 and both CYP71E7 paralogs in the cortex, xylem and phloem parenchyma, and in specific cells in the endodermis of the petiole of the first unfolded leaf. Based on the data obtained, UGT85K4 and UGT85K5 are concluded to be the UGTs catalyzing in planta synthesis of cyanogenic glucosides. The localization of the biosynthetic enzymes suggests that cyanogenic glucosides may play a role in both defense reactions and in fine-tuning nitrogen assimilation in cassava.
authors: Plant Biochemistry Laboratory, Department of Plant Biology and Biotechnology, Villum Foundation Research Centre "Pro-Active Plants", UNIK Center for Synthetic Biology, University of Copenhagen, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark.
authors: Rubini
authors: R
authors: Kannangara
authors: Mohammed S
authors: MS
authors: Motawia
authors: Natascha K K
authors: NK
authors: Hansen
authors: Suzanne M
authors: SM
authors: Paquette
authors: Carl E
authors: CE
authors: Olsen
authors: Birger L
authors: BL
authors: Møller
authors: Kirsten
authors: K
authors: Jørgensen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D055162:Biocatalysis; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D005960:Glucosides / Q000737:chemistry / Q000378:metabolism*; D005964:Glucosyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D002365:Manihot / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009570:Nitriles / Q000737:chemistry / Q000378:metabolism*; D010802:Phylogeny; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
chemical_list: D000906:Antibodies; D018076:DNA, Complementary; D005960:Glucosides; D009570:Nitriles; D010940:Plant Proteins; D011994:Recombinant Proteins; D005964:Glucosyltransferases; C586855:UGT85K4 protein, Manihot esculenta; C586856:UGT85K5 protein, Manihot esculenta; C005091:linamarin; C001556:lotaustralin