Molecular and Structural Characterization of a Promiscuous C‐Glycosyltransferase from Trollius chinensis

Abstract

Herein, the catalytic promiscuity of TcCGT1, anew C-glycosyltransferase (CGT) from the medicinal plant Trollius chinensis is explored. TcCGT1 could efficiently and regiospecifically catalyze the 8-C-glycosylation of 36 flavones and other flavonoids and could also catalyze the O-glycosylation of diverse phenolics.The crystal structure of TcCGT1 in complex with uridine diphosphate was determined at 1.85 resolution. Molecular docking revealed an ew model for the catalytic mechanism of TcCGT1, which is initiated by the spontaneous deprotonation of the substrate.T he spacious binding pocket explains the substrate promiscuity,and the binding pose of the substrate determines C-or O-glycosylation activity.S itedirected mutagenesis at two residues (I94E and G284K) switched C-to O-glycosylation. TcCGT1 is the first plant CGT with ac rystal structure and the first flavone 8-Cglycosyltransferase described. This provides abasis for designing efficient glycosylation biocatalysts.

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

“…Generally, most of the known GTs, including OGTs and CGTs, exhibit the highest catalytic activity at pH 7.0-9.0 and lower catalytic activity at pH >10.0 15,[23][24][25][26][27][28][29][30][31][32] . However, the maximum catalytic activity of AbCGT was observed at pH 11.0 (Supplementary Fig.…”

“…Acyl groups, as electron-donating and potential positioning groups, are necessary for the substrates of known plant CGTs 15,[23][24][25][26][27][28][29][30][31][32] . Among the substrates of acyl resorcinols, which should be both C-and O-glycosylated according to the above catalytic characteristics of AbCGT, there was an exception that 3,5-dihydroxyacetophenone (4) was only O-glycosylated (Supplementary Fig.…”

“…Then we designed and chemically synthesized 25 potential substrates . Functional groups, including aliphatic carbon chains (30)(31)(32)(33), saturated carbon cycles (34)(35)(36)(37), carbon chain-substituted benzenes (38)(39)(40)(41), and N-and S-heterocycles (42-45), were chemically attached to phloroglucinol by a one-step reaction 43 . Furthermore, various derivatives (46-54) of 1-benzylbenzene-2,4,6triol (38) and 1-phenethylbenzene-2,4,6-triol (39) were chemically synthesized to investigate the effects of different substituents in the B ring on the pharmacological activity.…”

“…OsCGT was the first flavonoid CGT identified from Oryza sativa, which initiated the exploration of plant CGTs. From 2009 to 2020, about ten plant CGTs were identified 15,[23][24][25][26][27][28][29][30][31] . Recently, a highly promiscuous CGT (GgCGT) with C-, O-, N-, and S-glycosylation activity was identified from Glycyrrhiza glabra 32 .…”

“…GgCGT could efficiently catalyze C-glycosylation of substrates containing a flopropione unit. However, these discovered CGTs function primarily on C-glycosylating flavonoids [23][24][25][26][27][28][29][30] and other phenols with acyl groups 15,31,32 . In our previous work, although the substrate scopes of the mutated variants of a Mangifera indica CGT were expanded, the structural diversity of substrates is still limited, for example, an acyl substituent of phenolic acceptors is necessary for C-glycosylation 31 .…”