UGT74AN1, a Permissive Glycosyltransferase from Asclepias curassavica for the Regiospecific Steroid 3-O-Glycosylation

Abstract

A permissive steroid glycosyltransferase (UGT74AN1) from Asclepias curassavica exhibited robust capabilities for the regiospecific C3 glycosylation of cardiotonic steroids and C steroid precursors, and unprecedented promiscuity toward 53 structurally diverse natural and unnatural compounds to form O-, N-, and S-glycosides, along with the catalytic reversibility for a one-pot transglycosylation reaction. These findings highlight UGT74AN1 as the first regiospecific catalyst for cardiotonic steroid C3 glycosylation and exhibit significant potential for glycosylation of diverse bioactive molecules in drug discovery.

An Efficient One‐Pot Enzymatic Synthesis of Cardiac Glycosides with Varied Sugar Chain Lengths

“…UGT74AN3 also exhibited catalytic promiscuity for the regiospecific C‐3 glycosylation of structurally diverse CTS with UDP‐glucose (Scheme , Table S3). In combination with previous studies, we could infer that GTs from the UGT74AN sub‐family are likely to possess the ability to catalyze the glycosylation of CTS (Figure S2), which supports the further mining of related functional enzymes in cardenolide biosynthesis. To the best of our knowledge, UGT74AN3 is the first steroid GT cloned from C roseus .…”

“…The structures were identified by HR‐ESI‐MS, 1 H NMR, 13 C NMR and HMBC spectroscopy (Figure S3, S24–S36). 1 a was confirmed as resibufogenin 3‐ O ‐β‐D‐glucoside by comparison with the published data . The HR‐ESI‐MS spectrum of 1 b showed a pseudo‐molecular ion [M+Na] + peak at m/z 731.3184, indicating a molecular formula of C 36 H 52 O 14 (calcd.…”

“…In the past few years, significant progress has been achieved in the discovery of GTs from microbes for CTS glycosylation, such as OleD and YjiC1 . Previously, we reported the glycosylation of CTS using a plant GT, UGT74AN1, which exhibited robust capabilities for the regiospecific C‐3 glycosylation of CTS . However, the GTs from both microbes and plants identified thus far can only generate mono‐ or di‐ O ‐glycosylation products .…”

“…Previously, we reported the glycosylation of CTS using a plant GT, UGT74AN1, which exhibited robust capabilities for the regiospecific C‐3 glycosylation of CTS . However, the GTs from both microbes and plants identified thus far can only generate mono‐ or di‐ O ‐glycosylation products . Enzymatic syntheses of cardiac glycosides with varied sugar chain lengths as safer cardiotonic agents has not yet been achieved, due to the lack of a suitable enzyme or chemical approach for the extension of the sugar chain .…”

“…In our previous work, we found a promiscuous plant steroid GT, UGT74AN1, from Asclepias curassavica . However, its instability and poor expression in E. coli inspired us to search for more powerful steroid GTs in other plants.…”