Characterization and expression profile of two UDP‐glucosyltransferases, UGT85K4 and UGT85K5, catalyzing the last step in cyanogenic glucoside biosynthesis in cassava

Abstract

SUMMARYManihot 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.

Volatile Glycosylation in Tea Plants: Sequential Glycosylations for the Biosynthesis of Aroma β-Primeverosides Are Catalyzed by Two Camellia sinensis Glycosyltransferases

“…7). CsGT1 showed high similarity to cassava (Manihot esculenta) UGT85K4 and UGT85K5 involved in the biosynthesis of cyanogenic glucosides (Kannangara et al, 2011). In contrast, CsGT2 constitutes a new member of the so-called sugarsugar UGT or glycoside-specific glycosyltransferase (GGT) group that specifically catalyzes glycosylation at the sugar moiety of various phytochemical glycosides (but not aglycones), including the morning glory DUSKY (UGT79G16) and tomato NSGT1 involved in the glucosylation of anthocyanin and volatile glycosides, respectively (Morita et al, 2005;Tikunov et al, 2013).…”

Comparative Analysis of Benzoxazinoid Biosynthesis in Monocots and Dicots: Independent Recruitment of Stabilization and Activation Functions

“…Like in benzoxazinoid biosynthesis, the branchpoint enzyme homospermidine synthase evolved by repeated independent recruitment of a primary function, deoxyhypusine synthase. The analysis of widespread plant secondary pathways, such as flavonoid and cyanogenic glucoside biosynthesis, demonstrated recruitment of genes from the same gene family for the respective enzymatic steps in the pathways, such as the CYP75 flavonoid hydroxylases in angiosperm flavonoid biosynthesis (Nelson and Werck-Reichhart, 2011), CYP79 and CYP71E monooxygenases , and UGT85 glucosyltransferases in cyanogenic glucoside biosynthesis (Jones et al, 1999;Franks et al, 2008;Kannangara et al, 2011). However, the recruited genes are not necessarily orthologous …”

A recycling pathway for cyanogenic glycosides evidenced by the comparative metabolic profiling in three cyanogenic plant species

“…Glucosyl transferases producing linamarin and lotaustralin (UGT85K4, UGT85K5) [60], prunasin A c c e p t e d M a n u s c r i p t (UGT85A19) [61] and dhurrin (UGT85B1) [62] have been described in cassava, almond and sorghum, respectively. Nothing is known about the glucosyltransferases and apiosyl-, xylosyl-and arabinosyltransferases which produce diglycosides in these plants.…”