Probing and Engineering Key Residues for Bis-<i>C</i>-glycosylation and Promiscuity of a <i>C</i>-Glycosyltransferase

Chen, Fan; Fan, Shuai; Chen, Ridao; Xie, Kebo; Yin, Sha; Sun, Lili; Liu, Jimei; Yang, Lin; Kong, Jian-Qiang; Yang, Zhaoyong; Dai, Jungui

doi:10.1021/acscatal.8b00376

Cited by 45 publications

(45 citation statements)

References 69 publications

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“…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.…”

Section: Resultsmentioning

confidence: 99%

“…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 .…”

mentioning

confidence: 99%

“…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 . Therefore, mining CGTs with catalytic stereospecificity and promiscuity toward other structurally different substrates and further establishing a green and economical biosynthesis route for bioactive C-glycosides are significant for drug design and discovery.…”

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confidence: 99%

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

et al. 2020

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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.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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

et al. 2020

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“…Up to now, several UGTs have been characterized in P. ginseng , which exhibit strict substrate selectivity and specificity [ 11 , 12 , 13 ]. It has been reported that UGTs from microbes possess a more open and larger acceptor binding pocket than those from plants and therefore endow themselves with broad substrate specificity [ 29 , 30 , 31 ]. Therefore, research on microbial UGTs has attracted extensive interest and made great progress in the enzymatic glycosylation of natural and unnatural products.…”

Section: Discussionmentioning

confidence: 99%

Enzymatic Synthesis of Unnatural Ginsenosides Using a Promiscuous UDP-Glucosyltransferase from Bacillus subtilis

Zhang

Gong

et al. 2018

Molecules

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Glycosylation, which is catalyzed by UDP-glycosyltransferases (UGTs), is an important biological modification for the structural and functional diversity of ginsenosides. In this study, the promiscuous UGT109A1 from Bacillus subtilis was used to synthesize unnatural ginsenosides from natural ginsenosides. UGT109A1 was heterologously expressed in Escherichia coli and then purified by Ni-NTA affinity chromatography. Ginsenosides Re, Rf, Rh1, and R1 were selected as the substrates to produce the corresponding derivatives by the recombinant UGT109A1. The results showed that UGT109A1 could transfer a glucosyl moiety to C3-OH of ginsenosides Re and R1, and C3-OH and C12-OH of ginsenosides Rf and Rh1, respectively, to produce unnatural ginsenosides 3,20-di-O-β-d-glucopyranosyl-6-O-[α-l-rhamnopyrano-(1→2)-β-d-glucopyranosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol (1), 3,20-di-O-β-d-glucopyranosyl-6-O-[β-d-xylopyranosyl-(1→2)-β-d-glucopyranosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol (6), 3-O-β-d-glucopyranosyl-6-O-[β-d-glucopyranosyl-(1→2)-β-d-glucopyranosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol (3), 3,12-di-O-β-d-glucopyranosyl-6-O-[β-d-glucopyranosyl-(1→2)-β-d-glucopyranosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol (2), 3,6-di-O-β-d-glucopyranosyl-dammar-24-ene-3β,6α,12β,20S-tetraol (5), and 3,6,12-tri-O-β-d-glucopyranosyl-dammar-24-ene-3β,6α,12β,20S-tetraol (4). Among the above products, 1, 2, 3, and 6 are new compounds. The maximal activity of UGT109A1 was achieved at the temperature of 40 °C, in the pH range of 8.0–10.0. The activity of UGT109A1 was considerably enhanced by Mg2+, Mn2+, and Ca2+, but was obviously reduced by Cu2+, Co2+, and Zn2+. The study demonstrated that UGT109A1 was effective in producing a series of unnatural ginsenosides through enzymatic reactions, which could pave a way to generate promising leads for new drug discovery.

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“…The purified UGT88E18 was applied to the in vitro reaction with calycosin (Compound 1) or formononetin (Compound 2, another important isoflavonoid present in R. astragali) as the Chemical glucosylation is notoriously complicated because of various disadvantages such as low efficiency, poor stereospecificity, a series of protection and deprotection steps, and environmental pollution [14]. In this respect, enzymatic glucosylation using regio-and stereoselective UGTs can alleviate these disadvantages [15,16]. Extensive studies indicate that sucrose synthase (SuSy) is a versatile biocatalyst that can synthesize costly uridine diphosphate glucose (UDPG) from abundant and cheap sucrose and catalytic amounts of uridine diphosphate (UDP) [17,18].…”

Section: Regioselective Glucosylation Of Calycosin and Formononetin Bmentioning

confidence: 99%

Biocatalytic Synthesis of Calycosin-7-O-β-D-Glucoside with Uridine Diphosphate–Glucose Regeneration System

Min

et al. 2020

Catalysts

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Calycosin-7-O-β-D-glucoside (Cy7G) is one of the principal components of Radix astragali. This isoflavonoid glucoside is regarded as an indicator to assess the quality of R. astragali and exhibits diverse pharmacological activities. In this study, uridine diphosphate-dependent glucosyltransferase (UGT) UGT88E18 was isolated from Glycine max and expressed in Escherichia coli. Recombinant UGT88E18 could selectively and effectively glucosylate the C7 hydroxyl group of calycosin to synthesize Cy7G. A one-pot reaction by coupling UGT88E18 to sucrose synthase (SuSy) from G. max was developed. The UGT88E18–SuSy cascade reaction could recycle the costly uridine diphosphate glucose (UDPG) from cheap sucrose and catalytic amounts of uridine diphosphate (UDP). The important factors for UGT88E18–SuSy cascade reaction, including UGT88E18/SuSy ratios, different temperatures, and pH values, different concentrations of dimethyl sulfoxide (DMSO), UDP, sucrose, and calycosin, were optimized. We produced 10.5 g L−1 Cy7G in the optimal reaction conditions by the stepwise addition of calycosin. The molar conversion of calycosin was 97.5%, with a space–time yield of 747 mg L−1 h−1 and a UDPG recycle of 78 times. The present study provides a new avenue for the efficient and cost-effective semisynthesis of Cy7G and other valuable isoflavonoid glucosides by UGT–SuSy cascade reaction.

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Probing and Engineering Key Residues for Bis-C-glycosylation and Promiscuity of a C-Glycosyltransferase

Cited by 45 publications

References 69 publications

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

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

Enzymatic Synthesis of Unnatural Ginsenosides Using a Promiscuous UDP-Glucosyltransferase from Bacillus subtilis

Biocatalytic Synthesis of Calycosin-7-O-β-D-Glucoside with Uridine Diphosphate–Glucose Regeneration System

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