- Functional Characterization and Protein Engineering of a Triterpene 3-/6-/2′-O-Glycosyltransferase Reveal a Conserved Residue Critical for the Regiospecificity
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Engineering the function of triterpene glucosyltransferases (GTs) is challenging due to the large size of the sugar acceptors. In this work, we identified a multifunctional glycosyltransferase AmGT8 catalyzing triterpene 3-/6-/2′-O-glycosylation from the medicinal plant Astragalus membranaceus. To engineer its regiospecificity, a small mutant library was built based on semi-rational design. Variants A394F, A394D, and T131V were found to catalyze specific 6-O, 3-O, and 2′-O glycosylation, respectively. The origin of regioselectivity of AmGT8 and its A394F variant was studied by molecular dynamics and hydrogen deuterium exchange mass spectrometry. Residue 394 is highly conserved as A/G and is critical for the regiospecificity of the C- and O-GTs TcCGT1 and GuGT10/14. Finally, astragalosides III and IV were synthesized by mutants A394F, T131V and P192E. This work reports biocatalysts for saponin synthesis and gives new insights into protein engineering of regioselectivity in plant GTs.
- Bao, Yang-Oujie,Gao, Bai-Han,Li, Fu-Dong,Qiao, Xue,Shi, Xiao-Meng,Su, Hui-Fei,Wang, Hai-Dong,Ye, Min,Yi, Yang,Zhang, Meng
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supporting information
(2022/01/06)
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- Efficient Production of Orientin and Vitexin from Luteolin and Apigenin Using Coupled Catalysis of Glycosyltransferase and Sucrose Synthase
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Orientin and vitexin are flavone 8-C-glycosides that exhibit many biological characteristics. This study aimed to establish a two-enzyme-coupled catalytic strategy to enhance the biosynthesis of orientin and vitexin from apigenin and luteolin, respectively. The C-glucosyltransferase (TcCGT1) gene from Trollius chinensis was cloned and expressed in Escherichia coli BL21(DE3). The optimal activity of TcCGT1 was achieved at pH 9.0 and 37 °C. TcCGT1 was relatively stable over the pH range of 7.0-10.0 at a temperature lower than 45 °C. The coupled catalytic strategy of TcCGT1 and different sucrose synthases was adopted to enhance the production of orientin and vitexin. By optimizing the coupling reaction conditions, orientin and vitexin production successfully achieved 2324.4 and 5524.1 mg/L with a yield of 91.4 and 89.3% (mol/mol), respectively. The coupled catalytic strategy proposed in this study might serve as a promising candidate for the large-scale production of orientin and vitexin in the future.
- Liu, Shike,Lyu, Yunbin,Yu, Shiqin,Cheng, Jie,Zhou, Jingwen
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p. 6578 - 6587
(2021/06/28)
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- Design and Synthesis of CNb-targeted Flavones and Analogues with Neuroprotective Potential Against H2O2- and Aβ1-42-Induced Toxicity in SH-SY5Y Human Neuroblastoma Cells
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With the lack of available drugs able to prevent the progression of Alzheimer’s disease (AD), the discovery of new neuroprotective treatments able to rescue neurons from cell injury is presently a matter of extreme importance and urgency. Here, we were inspired by the widely reported potential of natural flavonoids to build a library of novel flavones, chromen-4-ones and their C-glucosyl derivatives, and to explore their ability as neuroprotective agents with suitable pharmacokinetic profiles. All compounds were firstly evaluated in a parallel artificial membrane permeability assay (PAMPA) to assess their effective permeability across biological membranes, namely the blood-brain barrier (BBB). With this test, we aimed not only at assessing if our candidates would be well-distributed, but also at rationalizing the influence of the sugar moiety on the physicochemical properties. To complement our analvsis logD7.4 was determined. From all screened compounds, the p-morpholinyl flavones stood out for their ability to fully rescue SH-SY5Y human neuroblastoma cells against both H2O2 A β1-42-induced cell death. Cholinesterase inhibition was also evaluated, and modest inhibitory activities were found. This work highlights the potential of C-glucosylflavones as neuroprotective agents, and presents the p-morpholinyl C-glucosylflavone 37, which did not show any cytotoxicity towards HepG2 and Caco-2 cells at 100 ΜM, as a new lead structure for further development against AD.
- Matos, Ana M. de,Martins, Alice,Man, Teresa,Evans, David,Walter, Magnus,Oliveira, Maria Concei??o,López, óscar,Fernandez-Bola?os, José G.,D?twyler, Philipp,Ernst, Beat,Macedo, M. Paula,Contino, Marialessandra,Colabufo, Nicola A.,Rauter, Amélia P.
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- Molecular and Structural Characterization of a Promiscuous C-Glycosyltransferase from Trollius chinensis
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Herein, the catalytic promiscuity of TcCGT1, a new C-glycosyltransferase (CGT) from the medicinal plant Trollius chinensis is explored. TcCGT1 could efficiently and regio-specifically 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 a new model for the catalytic mechanism of TcCGT1, which is initiated by the spontaneous deprotonation of the substrate. The spacious binding pocket explains the substrate promiscuity, and the binding pose of the substrate determines C- or O-glycosylation activity. Site-directed mutagenesis at two residues (I94E and G284K) switched C- to O-glycosylation. TcCGT1 is the first plant CGT with a crystal structure and the first flavone 8-C-glycosyltransferase described. This provides a basis for designing efficient glycosylation biocatalysts.
- He, Jun-Bin,Zhao, Peng,Hu, Zhi-Min,Liu, Shuang,Kuang, Yi,Zhang, Meng,Li, Bin,Yun, Cai-Hong,Qiao, Xue,Ye, Min
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supporting information
p. 11513 - 11520
(2019/07/16)
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- Biosynthesis of natural and novel C-glycosylflavones utilising recombinant Oryza sativa C-glycosyltransferase (OsCGT) and Desmodium incanum root proteins
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The rice C-glycosyltransferase (OsCGT) is one of only a small number of characterised plant C-glycosyltransferases (CGT) known. The enzyme C-glucosylates a 2-hydroxyflavanone substrate with UDP-glucose as the sugar donor to produce C-glucosyl-2-hydroxyflavanones. We tested substrate specificity of the enzyme, using synthetic 2-hydroxyflavanones, and showed it has the potential to generate known natural CGFs that have been isolated from rice and also other plants. In addition, we synthesised novel, unnatural 2-hydroxyflavanone substrates to test the B-ring chemical space of substrate accepted by the OsCGT and demonstrated the OsCGT capacity as a synthetic reagent to generate significant quantities of known and novel CGFs. Many B-ring analogues are tolerated within a confined steric limit. Finally the OsCGT was used to generate novel mono-C-glucosyl-2-hydroxyflavanones as putative biosynthetic intermediates to examine the potential of Desmodium incanum biosynthetic CGTs to produce novel di-C-glycosylflavones, compounds implicated in the allelopathic biological activity of Desmodium against parasitic weeds from the Striga genus.
- Hao,Caulfield,Hamilton,Pickett,Midega,Khan,Wang,Hooper
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- Flavone C-glycosides from the flowers of Trollius chinensis and their anti-complementary activity
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Phytochemical investigation of ethanol extract from the flowers of Trollius chinensis Bunge resulted in the isolation of two new flavone C-glycosides (1-2), along with 10 known compounds (3-12). The structures of the new compounds were established as 6?-(
- Liu, Jiang-Yun,Li, Sheng-Yin,Feng, Jian-Yong,Sun, Yan,Cai, Jin-Na,Sun, Xiao-Fei,Yang, Shi-Lin
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p. 325 - 331
(2013/06/27)
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- PROCESS FOR PRODUCTION OF ORALLY INGESTIBLE COMPOSITION CONTAINING ARABINOFURANOSYL VITEXIN, AND USE OF THE COMPOSITION
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A method for producing an orally ingestible composition containing an arabinofuranosyl vitexin and/or a hydrolysate thereof by using Basella alba as a raw material. The product of such method has a sedative effect and a sleep-inducing effect.
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Page/Page column 6
(2012/01/14)
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- Glucose uptake enhancing activity of puerarin and the role of C-glucoside suggested from activity of related compounds
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Chemical treatment of diabetes mellitus is widely studied and controlling of blood glucose level is the main course of therapy. In type 2 diabetes mellitus, insulin resistance is the major problem. An isoflavone C-glucoside, puerarin (1), is known to enhance glucose uptake into the insulin sensitive cell and is thought to be a candidate for treatment of diabetes mellitus. We synthesized 1 and several derivatives to apply for the structure-activity relationship study. The result against 3T3-L1 adipocyte indicated that the C-glucoside part of 1 is unconcerned in its activity when tested in vitro and the main structure responsible for its activity was the isoflavone moiety.
- Kato, Eisuke,Kawabata, Jun
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supporting information; experimental part
p. 4333 - 4336
(2010/09/18)
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- Quantitation of chafurosides A and B in tea leaves and isolation of prechafurosides A and B from oolong tea leaves
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A procedure was developed for the quantitative determination of chafuroside A, a flavone C-glycoside with potent anti-inflammatory activity, and its regioisomer chafuroside B, as well as isovitexin and vitexin, by selected reaction monitoring liquid chromatography-tandem mass spectrometry (SRM LC-MS/MS) analysis. This method was successfully applied to commercial leaves of green tea, houji tea, oolong tea, and black tea. High levels of chafurosides A and B were found in oolong tea leaves that had been heated at >140°C. Next, their precursors, prechafurosides A and B, were isolated from methanol extract of oolong tea leaves prepared from Shizu 7132, Camellia sinensis (L.) O. Kuntze, by partition with n-butanol and H2O and chromatography on Diaion SP-825, Sephadex LH-20, and ODS C-18, guided by assay of chafuroside formation. Prechafurosides A and B gave chafurosides A and B, respectively, in good yields when heated at 160°C for 0.5 h. Solvolysis of prechafurosides A and B with pyridine and dioxane quantitatively afforded isovitexin and vitexin, respectively. On the basis of these results and physicochemical data (MS, UV, and NMR), prechafurosides A and B were concluded to be new flavone C-glycoside sulfates, isovitexin2"-sulfate and vitexin-2″-sulfate, respectively.
- Ishida, Hitoshi,Wakimoto, Toshiyuki,Kitao, Yukiko,Tanaka, Shimako,Mlyase, Toshio,Nukaya, Haruo
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scheme or table
p. 6779 - 6786
(2010/08/19)
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- Concise synthesis of chafurosides A and B
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The regioselective synthesis of chafurosides A (1) and B (2) from the same methyl ketone 5 was accomplished using a novel protecting group strategy. Both flavone rings were constructed from β-diketone intermediate 4, which was readily obtained by condensation of an acyl donor and ketone 5. Construction of the dihydrofuran ring was achieved via an intramolecular Mitsunobu reaction.
- Furuta, Takumi,Nakayama, Miho,Suzuki, Hirotaka,Tajimi, Hiroko,Inai, Makoto,Nukaya, Haruo,Wakimoto, Toshiyuki,Kan, Toshiyuki
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supporting information; experimental part
p. 2233 - 2236
(2009/10/02)
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- Glycosyl Imidates, 69. - Synthesis of Flavone C-Glycosides Vitexin, Isovitexin, and Isoembigenin
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2-Hydroxy-4,6-dimethoxyacetophenone (4) was glycosylated with O-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl) trichloroacetimidate (5) and trimethylsilyl triflate as promoter to yield directly the C-glycoside 6.Construction of the flavone system by application of a Baker-Venkataraman-type rearrangement followed by deprotection yielded isoembigenin (2).Glycosylation of 4,6-bis(tert-butyldimethylsilyloxy)-2-hydroxyacetophenone (17) with the trichloroacetimidate 5 afforded the O-glycoside intermediate 18 which was converted via Fries rearrangement into the C-glycoside 21.Applying again the Baker-Venkataraman rearrangement and cyclization gave isovitexin and vitexin derivatives 25 and 26, which were completely deprotected to yield isovitexin (1b) and vitexin (1a), respectively. - Key Words: Glycosides / Flavones / Vitexin / Isovitexin / Isoembigenin / Carbohydrates / Trichloroacetimidates / Fries rearrangement / Baker-Venkataraman rearrangement
- Mahling, Juergen-Andreas,Jung, Karl-Heinz,Schmidt, Richard R.
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p. 461 - 466
(2007/10/02)
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