A novel glucuronosyltransferase has an unprecedented ability to catalyse continuous two‐step glucuronosylation of glycyrrhetinic acid to yield glycyrrhizin

Abstract

Glycyrrhizin is an important bioactive compound that is used clinically to treat chronic hepatitis and is also used as a sweetener world-wide. However, the key UDP-dependent glucuronosyltransferases (UGATs) involved in the biosynthesis of glycyrrhizin remain unknown. To discover unknown UGATs, we fully annotated potential UGATs from Glycyrrhiza uralensis using deep transcriptome sequencing. The catalytic functions of candidate UGATs were determined by an in vitro enzyme assay. Systematically screening 434 potential UGATs, we unexpectedly found one unique GuUGAT that was able to catalyse the glucuronosylation of glycyrrhetinic acid to directly yield glycyrrhizin via continuous two-step glucuronosylation. Expression analysis further confirmed the key role of GuUGAT in the biosynthesis of glycyrrhizin. Site-directed mutagenesis revealed that Gln-352 may be important for the initial step of glucuronosylation, and His-22, Trp-370, Glu-375 and Gln-392 may be important residues for the second step of glucuronosylation. Notably, the ability of GuUGAT to catalyse a continuous two-step glucuronosylation reaction was determined to be unprecedented among known glycosyltransferases of bioactive plant natural products. Our findings increase the understanding of traditional glycosyltransferases and pave the way for the complete biosynthesis of glycyrrhizin.

Multi‐tissue integration of transcriptomic and specialized metabolite profiling provides tools for assessing the common bean (Phaseolus vulgaris) metabolome

“…A cluster with soybean's GmUGT73F2 and GmUGT73F4 catalyzing the addition of glucose and xylose, respectively, to an arabinose residue at the C‐22 position of soyasaponins from the A class (Sayama et al ., ) and Medicago 's UGT73F3 involved in the glucosylation of multiple sapogenins at position C‐28 (Naoumkina et al ., ) also included the bean candidate Phvul.003G097300. The last cluster of UGTs involved in triterpenoid biosynthesis including candidates from beans was formed by G. uralensis GuUGAT involved in a two‐step glucuronosylation yielding glycyrrhizin (Xu et al ., ) and three candidates localized in tandem in the same region of chromosome 3 (Phvul.003G046900, Phvul.003G047000 and Phvul.003G047100). Finally, no candidates were identified close to glycoalkaloid‐related proteins, and a group of three candidates (Phvul.008G090500, Phvul.008G090600 and Phvul.008G090900) was localized inbetween the clade of GmSGT2 and GuUGAT.…”

“…Common bean has four copies from which only Phvul.010G101800 and Phvul.010G101600 are expressed in the investigated tissues, at particularly higher levels in the seedlings. A cluster with soybean's GmUGT73F2 and GmUGT73F4 catalyzing the addition of glucose and xylose, respectively, to an arabinose residue at the C-22 position of soyasaponins from the A class (Sayama et al, 2012) and Medicago's UGT73F3 involved in the glucosylation of multiple sapogenins at position C-28 (Naoumkina et al, 2010) triterpenoid biosynthesis including candidates from beans was formed by G. uralensis GuUGAT involved in a twostep glucuronosylation yielding glycyrrhizin (Xu et al, 2016) and three candidates localized in tandem in the same region of chromosome 3 (Phvul.003G046900, Phvul. 003G047000 and Phvul.003G047100).…”

“…Most of the knowledge regarding the biosynthesis and regulation of these compounds comes from studies in the model legume M. truncatula (Achnine et al, 2005;Naoumkina et al, 2010;Carelli et al, 2011;Fukushima et al, 2011Fukushima et al, , 2013Pollier et al, 2011;Biazzi et al, 2015;Mertens et al, 2015). Nevertheless, many genes, particularly UDP-sugar dependent glycosyltransferase (UGTs) and cytochrome P450 monooxygenases (CYPs) were also functionally characterized in several plants with economical relevance and interesting medicinal properties, such as soybean (Shibuya et al, 2006(Shibuya et al, , 2010Sayama et al, 2012) and Glycyrrhiza uralensis (Seki et al, 2008;Xu et al, 2016;Tamura et al, 2017). A great resource for plant triterpene biosynthesis including structures, enzymes and pathways in multiple plants was recently made available through the TriForC database (Miettinen et al, 2018).…”

Functional specialization of UDP‐glycosyltransferase 73P12 in licorice to produce a sweet triterpenoid saponin, glycyrrhizin

“…Recently, Xu et al . reported that GuUGAT, which corresponds to UGT73B27 in our study but differs from UGT73B27 by one amino acid residue (Leu329 in GuUGAT versus Phe329 in UGT73B27), has catalytic activity to produce glycyrrhizin directly from glycyrrhetinic acid through a continuous two‐step glucuronosylation reaction (Xu et al ., ). The target enzymatic activity was, however, not detected in UGT73B27 in our assay (Figure S9).…”

Wide Identification of the Compounds in Licorice and Exploration of the Mechanism for Prostatitis Treatment by Combining UHPLC‐LTQ‐Orbitrap MS with Network Pharmacology

“…Accordingly, many kinds of licorice products had been developed in recent years , . It was reported that famous magnesium isoglycyrrhizinate injection, developed from licorice, reached sales of CNY ¥1.6 billion in China in 2014 . Interestingly, recent study showed that extracts from licorice also had good pharmacological roles in treating prostatitis .…”