Analysis of Two New Arabinosyltransferases Belonging to the Carbohydrate-Active Enzyme (CAZY) Glycosyl Transferase Family1 Provides Insights into Disease Resistance and Sugar Donor Specificity

Abstract

Glycosylation of small molecules is critical for numerous biological processes in plants, including hormone homeostasis, neutralization of xenobiotics, and synthesis and storage of specialized metabolites. Glycosylation of plant natural products is usually performed by uridine diphosphate-dependent glycosyltransferases (UGTs). Triterpene glycosides (saponins) are a large family of plant natural products that determine important agronomic traits such as disease resistance and flavor and have numerous pharmaceutical applications. Most characterized plant natural product UGTs are glucosyltransferases, and little is known about enzymes that add other sugars. Here we report the discovery and characterization of AsAAT1 (UGT99D1), which is required for biosynthesis of the antifungal saponin avenacin A-1 in oat (Avena strigosa). This enzyme adds L-Ara to the triterpene scaffold at the C-3 position, a modification critical for disease resistance. The only previously reported plant natural product arabinosyltransferase is a flavonoid arabinosyltransferase from Arabidopsis (Arabidopsis thaliana). We show that AsAAT1 has high specificity for UDP-b-L-arabinopyranose, identify two amino acids required for sugar donor specificity, and through targeted mutagenesis convert AsAAT1 into a glucosyltransferase. We further identify a second arabinosyltransferase potentially implicated in the biosynthesis of saponins that determine bitterness in soybean (Glycine max). Our investigations suggest independent evolution of UDP-Ara sugar donor specificity in arabinosyltransferases in monocots and eudicots.

Pathway-specific enzymes from bamboo and crop leaves biosynthesize anti-nociceptive C-glycosylated flavones

“…Carabinosides are unique to the leaves of grass family plants. Arabinosyltransferases of plant origin have been reported to be involved in the arabinoxylan biosynthesis [45][46][47] and in posttranslational modification of glycoproteins 48 , but UDParabinosyltransferases decorating secondary metabolites are hardly reported with just two cases 49,50 (identified as O-arabinosyltransferases). Recent genetic-based studies on rice and maize metabolism have implied some gene candidates for Carabinosylation [51][52][53][54] , but none have been functionally characterized to date.…”

Reconstruction of the Evolutionary Histories of UGT Gene Superfamily in Legumes Clarifies the Functional Divergence of Duplicates in Specialized Metabolism

“…D18 of G. max included two members (Glyma.08G348500 and Glyma.08G348600). One of these members (Glyma.08G348500) was co-expressed highly with UGT73F2 and characterized to attach arabinose at the C-22-O position of SB-glycoside in vitro [23]. The resulting product was subsequently utilized by UGT73F2 which attaches glucose as the second sugar to the C22-O-arabinose of SB in in vitro.…”

Plant terpenes that mediate below‐ground interactions: prospects for bioengineering terpenoids for plant protection

“…[53][54][55] These include genes that encode an oxidosqualene cyclase (saponin deficient 1, SAD1) that synthesises the -amyrin scaffold, 53 a multifunctional CYP(SAD2) that oxidizes -amyrin to install a C 12− C 13 epoxide and a C 16 hydroxyl group, 54 another CYP (SAD6) that introduces the C 21 hydroxyl group, 56 a methyl transferase (SAD9) that methylates anthranilate, 55 a glucosyltransferase UGT74H5 (SAD10) that glucosylates N-methyl anthranilate, 51 a serine carboxypeptidase-like acyltransferase (SCPL, SAD7) that acylates the deacyl avenacins, 55 and an arabinosyltransferase (UGT99D1) that catalyses the addition of an L-arabinose to the triterpene scaffold at the C3 position. 57 The biosynthesis of ginsenosides is under extensive investigation due to the medicinal properties of these compounds. Many genes involved in the biosynthesis of various ginsenosides have been identified, although the complete biosynthetic pathways are yet to be fully elucidated.…”