Exploring and applying the substrate promiscuity of a C-glycosyltransferase in the chemo-enzymatic synthesis of bioactive C-glycosides

Abstract

Bioactive natural C-glycosides are rare and chemical C-glycosylation faces challenges while enzymatic C-glycosylation catalyzed by C-glycosyltransferases provides an alternative way. However, only a small number of C-glycosyltransferases have been found, and most of the discovered C-glycosyltransferases prefer to glycosylate phenols with an acyl side chain. Here, a promiscuous C-glycosyltransferase, AbCGT, which is capable of C-glycosylating scaffolds lacking acyl groups, is identified from Aloe barbadensis. Based on the substrate promiscuity of AbCGT, 16 C-glycosides with inhibitory activity against sodium-dependent glucose transporters 2 are chemo-enzymatically synthesized. The C-glycoside 46a shows hypoglycemic activity in diabetic mice and is biosynthesized with a cumulative yield on the 3.95 g L‒1 scale. In addition, the key residues involved in the catalytic selectivity of AbCGT are explored. These findings suggest that AbCGT is a powerful tool in the synthesis of lead compounds for drug discovery and an example for engineering the catalytic selectivity of C-glycosyltransferases.

Exploring the catalytic function and active sites of a novel C-glycosyltransferase from Anemarrhena asphodeloides

“…2 C), indicating that 1a was a C -glycoside. The C -glycosylation can be distinguished from other types of glycosylation by the characteristic MS/MS fragment ions [M-H-120] − and [M-H-90] − , while that of O -, S -, and N -glucoside is [M-H-162] − /[M+H-162] + [ 23 ]. The structure of product 1a was further identified as 3- C- glucosylmaclurin by comparing with the glycosylated product of the reported enzyme MiCGT ( Fig.…”

“…In addition to flavonoid substrates, 3,4-dichlorobenzenethiol ( 3 ) could also be accepted by AaCGT. For S -glycosylation, the signature MS/MS fragment also appears at [M+H-162] + [ 23 ]. Based on ESI-MS analysis, together with the MS/MS spectrum that the product 3a showed the characteristic MS/MS fragment ion at m/z 176.9330 [M-H-162] − , AaCGT was found to be able to convert 3 into the corresponding S -glycoside at a close to 100% conversion rate ( Fig.…”

“…MiCGTb, the other efficient benzophenone CGT from Mangifera indica , also showed potent catalytic activities toward various benzophenone aglycones [ 21 ]. Besides, three other plant CGTs, such as FcCGT from Fortunella crassifolia [ 22 ], CuCGT from Citrus unshiu [ 22 ]and AbCGT from Aloe barbadensis [ 23 ], also displayed the C -glycosylation activities toward the benzophenone aglycone (maclurin) due to their possessing substrate promiscuities, but they are not generally recognized as benzophenone CGTs for the reason that maclurin is not their endogenous substrate, and the conversion rates that FcCGT and CuCGT catalyzed C -glycosylation of maclurin were rather low [ 22 ]. It is particularly noteworthy that the active sites for C -glycosylation of benzophenone CGTs, especially for determining interconversion of diverse types of glycosides, remain elusive.…”

Recent trends in biocatalysis

“…With dihydrochalcones as substrates, di- C -glycosylation can occur at the C6 and C8 positions. 226 Remarkably, a promiscuous C -glycosyltransferase from Trollius chinensis can accept multiple structures of flavones, flavonols, flavanones, flavanonols and dihydrochalcones, and introduce a glycosyl moiety at the C8 position. Meanwhile, it showed O -glycosylation activity on the C7 position when the C8 is already substituted by methoxyl or prenyl moieties.…”

Synthetic biology of plant natural products: From pathway elucidation to engineered biosynthesis in plant cells

“…Hence, it is possible to identify candidate genes by comparative analysis of samples with different metabolite compositions in target pathways; these samples may be from different plant species, accessions, or mutants, or from plants induced by a target-pathway stimulus. Differential transcriptional analysis can be performed on different samples to obtain candidate genes and enable further functional analysis ( Winzer et al., 2012 ; Galanie et al., 2015 ; Lau and Sattely, 2015 ; Polturak et al., 2016 ; Farrow et al., 2018 ; Kim et al., 2018 ; Christ et al., 2019 ; Yang et al., 2019 ; Nett et al., 2020 ; Stander et al., 2020 ; Xie et al., 2020 ).…”