Cyclocarya paliurus, (Batalin) Iljinsk., (Batalin) Iljinsk.

Li, Jie, Liu, Xiao, Gao, Yanrong, Zong, Guangning, Wang, Dandan, Liu, Meizi, Fei, Shang, Wei, Yu, Yin, Zhongping, Chen, Jiguang, Wang, Xiaoqiang & Shen, Yuequan, 2019, Identification of a UDP-Glucosyltransferase favouring substrate- and regiospecific biosynthesis of flavonoid glucosides in Cyclocarya paliurus, Phytochemistry 163, pp. 75-88 : 82-84

2.4. N- and S-glucosylation activity of C. paliurus GT1

In addition, to investigate the O -glucosylation activity of C. paliurus GT 1, we also explored whether C. paliurus GT 1 could catalyse S - and/or N -glucosylation on thiol (S)- and amine (N) sites of phenolic/aliphatic aglycones, with UDP-Glc as the donor substrate. Interestingly, C. paliurus GT 1 exhibited N - and S -glucosylation activities towards the simple aromatic molecules 3,4-dichloroaniline (DCA) and 4-chlorothiophenol (CTP), respectively, with high conversion rates (> 95%). In the HPLC analysis of CTP glucosylation catalysed by C. paliurus GT 1, a peak other than the monosaccharide product (CTPG) was speculated to be a disaccharide product (CTP2G), according to the molecular weight detected by LC-MS. However, no activity was observed with aliphatic molecules 4-chlorophenethylamine (CPEA) and 4-chlorobenzylmercaptan (CBM) ( Fig. 7 ). Control reactions lacking C. paliurus GT 1 confirmed that the reactions were enzyme-dependent, indicating that C. paliurus GT 1 has broad aglycon promiscuity.

Thus far, only a few N - and S -UGTs have been reported, e.g., Arabidopsis UGT 72B1 with both O - and N-glycosylation activities ( Brazier-Hicks et al., 2007), UGT74B1 with S-glycosylation function ( Grubb et al., 2014), and Bacillus cereus BcGT 1 with O -, S -, and N -glucosidation ( Chiu et al., 2016). Remarkably, C. paliurus GT 1 showed S - and N -glucosylation activities to catalyse the attachment of sugars to the aromatic amine or thiophenol groups, leading to the generation of N - or S -glucosides. However, C. paliurus GT 1 could not catalyse glucosylation on the amino or thiol group of the aliphatic chain towards formation of the corresponding glucosides. We hypothesize that C. paliurus GT 1 catalyses N - or S -glucosylation by utilizing a SN2-like reaction mechanism similar to its O -glucosylation and that it also has certain selectivity and energy requirements ( Hans et al., 2004). The oxygen atom of the phenolic, the sulphur atom of the thiophenol, and the nitrogen atom of the aromatic amine all contained a lone-pair of electrons and were activated by the aromatic group. Thereby the hydrogen atoms on the heteroatom were easily deprotonated by His-16 through direct and orientated nucleophilic attack compared with the corresponding atoms on an aliphatic chain. There is currently no additional evidence on C. paliurus GT 1-mediated glucosylation towards the disaccharide product of S -glucoside, or whether C. paliurus GT 1 can catalyse the formation of sugar chains. Further biochemical and mechanistic studies are needed to understand its multiple functions, especially the N - and S -glucosylation mechanisms.

2.5. Homology modelling and molecular docking of the C. paliurus GT 1

Due to the high degree of sequence similarity between C. paliurus GT 1 and UGT71G1 and the significant difference in the catalytic activity of glucosylation, it is necessary to understand the structural differences between the two proteins and to identify the key amino acid residue responsible for their differences. The homology-based structural model of C. paliurus GT 1 was constructed with the SWISS-MODEL server (https://swissmodel.expasy.org), using the crystal structure of UGT71G1 (PDB ID: 2ACW) as a template to predict the key amino acids involved in glucosylation. C. paliurus GT 1 consisted of similar N- and Cterminal domains with an α/β/α fold, and the sugar donor and acceptor binding pockets are located in a cleft between these two domains ( Fig. 8A ).

Recent studies have revealed important correlations between the amino acids around the binding pocket and its catalytic activity and selectivity, based on the crystal structural studies of some UGTs ( Fan et al., 2018; Modolo et al., 2009a, 2009b; Wilson et al., 2018). The homology-based structural modelling and docking studies provide some insights into the catalytic properties of C. paliurus GT 1. Molecular docking was performed to investigate the binding mode between quercetin and the enzymes. The docking results showed that the substrate binding pockets of C. paliurus GT 1 and UGT71G1 were similar; however, there were certain differences between C. paliurus GT 1 and UGT71G 1 in the binding site, which might be responsible for their differences in catalytic selectivity and activity. There are twenty key residues in the acceptor substrate quercetin binding pocket of the C. paliurus GT 1 structural model, six of which are conserved in UGT71G1; the rest differ from those in UGT71G1 ( Table 3 ).

In the docking model, UDP-glucose was almost completely buried in a long and narrow channel formed mainly by the PSPG motif in the Cterminal domain of C. paliurus GT 1. The 3’-, 4′- and 6′-OH groups of the glucose moiety formed hydrogen bonds with the OE2 atom of Q387, the N atom of W366, and the OG1 atom of T142 to further stabilize the transition states. These interactions were similar to the binding pattern of UGT71G1. Quercetin adopted a compact conformation to bind in the active site of C. paliurus GT 1. The phenyl B ring of quercetin was located at the hydrophobic pocket, surrounded by residues M121, F122, F147, F190, and A386, forming strong hydrophobic binding. A detailed analysis showed that the phenyl B ring of quercetin formed anion-π and π- π interactions with the side chains of residues E85 and F122, respectively. Importantly, the hydroxyl group at the 3′-position of the quercetin was close to the glucose moiety and C1′ atom of UDP-Glc, facilitating nucleophilic attack. All these interactions facilitated the anchoring of 3′- OH of quercetin in the active site and provided the best fitness score. UGT71G1 is capable of transferring glucose to each of the five hydroxyl groups on the quercetin molecule since all of them can dock close to the catalytic residue H22 within approximately 3 Å. We found that only the hydroxyl groups at the 3′- and 3-position could contact the corresponding catalytic residue H16 within approximately 3 Å. An acidic residue D120 was identified that may form an electron transfer chain with H16 to help deprotonate acceptor molecules, which is highly conserved in most plant UGTs ( Fig. 8B ).

The molecular surfaces of the two UGTs also exhibit differences. The W283 residue of the C. paliurus GT 1 may serve as a guard at the entrance of the substrate binding pocket. The large indole ring of W283 has strong hydrophobicity and steric hindrance compared with the corresponding residue M 286 in UGT71G1, and it may intercept large sugar acceptors, e.g., triterpenes. Moreover, compared with UGT71G1, an extra β- sheet (V309 to D320) in C. paliurus GT 1 forms a cap at the entrance of the substrate binding pocket, further reducing the size of the entrance. This modelling study provides some insights into the strict selectivity of C. paliurus of GTI for substrates ( Fig. 8C ).

Figures

Fig. 7. NGT and SGT activities of C. paliurus GT1 with N- and S-conjugating aglycones. HPLC chromatogram and MS analyses of the enzymatic reactions with C. paliurus GT1 in vitro. The UDP-glucose (UDPG) and N- and S- phenolic/aliphatic aglycons were the sugar donor and acceptor, respectively. The substrates and corresponding products were detected using HPLC chromatogram and MS.

Fig. 8. Comparison of the binding pattern of UDP- Glc (yellow sticks) and acceptor quercetin (black sticks) in C. paliurus GT1 (left) or UGT71G1 (right). (A) Homology model of C. paliurus GT1 or crystal structure of UGT71G1 docked with UDP-Glc and quercetin. (B) The substrate binding pocket in C. paliurus GT1 or UGT71G1 with the docked quercetin and the surrounding residues. (C) Molecular surfaces of C. paliurus GT1 or UGT71G1 showing their substrate binding pockets and entrance of the pockets. Residue W283 in C. paliurus GT1 or M286 in UGT71G1 is shown in red; residues V309 to D320 of the cap region in C. paliurus GT1 or the corresponding site (N313 to A315) in UGT71G1 are shown in green, respectively. White circles mark the entrance to the substrate binding pocket. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)