Enzymatic C-glycosylation: Insights from the study of a complementary pair of plant O- and C-glucosyltransferases

Abstract

C-Glycosylation presents a rare mode of sugar attachment to the core structure of natural products and is catalyzed by a special type of Leloir C-glycosyltransferases (C-GTs). Elucidation of mechanistic principles for these glycosyltransferases (GTs) is of fundamental interest, and it could also contribute to the development of new biocatalysts for the synthesis of valuable C-glycosides, potentially serving as analogues of the highly hydrolysis-sensitive O-glycosides. Enzymatic glucosylation of the natural dihydrochalcone phloretin from UDP-D-glucose was applied as a model reaction in the study of a structurally and functionally homologous pair of plant glucosyltransferases, where the enzyme from rice (Oryza sativa) was specific for C-glycosylation and the enzyme from pear (Pyrus communis) was specific for O-glycosylation. We show that distinct active-site motifs are used by the two enzymes to differentiate between C-and O-glucosylation of the phloretin acceptor. An enzyme design concept is therefore developed where exchange of active-site motifs results in a reversible switch between C/O-glycosyltransferase (C/O-GT) activity. Mechanistic proposal for enzymatic C-glycosylation involves a single nucleophilic displacement at the glucosyl anomeric carbon, proceeding through an oxocarbenium ion-like transition state. Alternatively, the reaction could be described as Friedel-Crafts-like direct alkylation of the phenolic acceptor.

Identification and functional analysis of 2‐hydroxyflavanone C‐glucosyltransferase in soybean (Glycine max)

“…Although the highly conserved N‐terminal histidine has been suggested to serve as a base to deprotonate the hydroxyl group of the acceptor substrate in OGT [15], the role of the corresponding residue in CGT is uncertain. Gutmann and Nidetzky have proposed that the N‐terminal histidine of Os CGT functions to deprotonate the phenolic hydroxyl group of the aromatic substrate, leading to activation of the aromatic carbon of the substrate by resonance [33]; however, the histidine residue may be insufficient for activating the aromatic ring compared with the anionic residue. The present study suggests a role for the N‐terminal histidine that is different from the one they proposed.…”

Enzymatic synthesis of glycosides: from natural O- and N-glycosides to rare C- and S-glycosides

“…At least 5 families of aromatic aglycones have been reported to be C -glycosylated: flavones, xanthones, chromones, anthrones, and gallic acids. Several corresponding plant C -GT have been cloned, expressed and characterized, from several crops including maize [ 35 ], rice [ 36 – 37 ], wheat [ 36 ], buckwheat [ 38 ] and other plants such as Arabidopsis [ 39 ] or Mangifera indica [ 40 ] ( Fig. 4 ).…”

“…Bacterial C -GTs are the last group identified in Salmonella enterica and Escherichia coli [ 44 – 46 ] that are involved in the biosynthesis of siderophores, that were shown to be C -glycosylated enterobactins. In addition to these naturally occurring C -GTs, engineering of O -GT to C -GT were successfully performed in several studies [ 37 , 47 – 48 ], and chemoenzymatic syntheses of C -glycosides were described in other publications [ 40 , 49 – 51 ]. In all described C -GTs, the aglycone acceptor was found to be a derivative of polyhydroxybenzaldehyde, that exhibit an acidic carbon on the aromatic ring.…”

“…Depending on the nature of the substrate and the C -GT involved in the enzymatic reaction, several regioselectivities were observed. A mechanistic study in 2013 by Gutmann and Nidetzky demonstrated that C -glycosylation occurred through a direct nucleophilic attack of an acidic carbon, and showed evidence against an O -glycosylation followed by an O -to- C rearrangement [ 37 ]. A last family of C -GT are the enzymes involved in C -mannosylation of protein tryptophanes [ 37 ].…”

Biocatalyzed synthesis of antidiabetic drugs: A review

“…Any biocatalytic method for creating the C-glycoside would demand the use of Leloir C-glycosiltransferases (C-GTs) (Gutmann and Nidetzky 2013), but this possibility has not been developed for gliflozins, as far as we know. Nevertheless, empagliflozin 98 does contain a chiral fragment in its structure, (S)tetrahydrofuran-3-ol (S)-103, required for the synthesis of the drug , and different biotransformations can be found in literature for producing both enantiomers of 103, as shown in Figure 33.…”