Structural Insights into the Substrate Recognition of Ginsenoside Glycosyltransferase Pq3‐O‐UGT2

Advanced Science, Год журнала: 2025, Номер unknown

Опубликована: Янв. 29, 2025

Abstract Ginsenosides are a group of tetracyclic triterpenoids with promising health benefits, consisting ginseng aglycone attached to various glycans. Pq3‐O‐UGT2, an important UDP‐dependent glycosyltransferase (UGT), catalyzes the production Ginsenoside Rg3 and Rd by extending glycan chain Rh2 F2, respectively, higher selectivity for F2. However, mechanism underlying its substrate recognition remains unclear. In this study, crystal structures Pq3‐O‐UGT2 in complex acceptor substrates solved. The revealed Nα5‐oriented binding pocket shaped unique conformation Nα5‐Nα6 linker. Hydrophobic interactions play pivotal role both while hydrogen bonds specifically aid F2 due additional glucose moiety. hydrophobic nature also enables recognize flavonoids. Overall, study provides novel insights into mechanisms ginsenoside UGTs, advancing understanding their function specificity.

Язык: Английский

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Functional Characterization and Protein Engineering of a Glycosyltransferase GcCGT to Produce Flavone 6,8-Di-C- and 6-C-4′-O-Glycosides

ACS Catalysis, Год журнала: 2024, Номер 14(2), С. 1075 - 1082

Опубликована: Янв. 8, 2024

Herein, we discovered an efficient flavone 6-C-glycosyltransferase GcCGT from the medicinal plant Gentiana crassicaulis. could catalyze consecutive two-step 6-C/4′-O-glycosylation of flavonoids. Homology modeling and site-directed mutagenesis yielded mutant F387K, which unprecedented 6-C-glycosylation 8-C-glycosides to produce 6,8-di-C-glycosides. To elucidate catalytic mechanisms, crystal structures GcCGT-apo (2.10 Å) GcCGT/UDP (2.40 were resolved. Structural analysis molecular dynamics simulations indicated that lack π–π stacking interaction for F387 changed protein conformation expanded entrance substrate binding pocket. This work provided method synthesize 6,8-di-C- 6-C-4′-O-glycosides.

Язык: Английский

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Construct Phenylethanoid Glycosides Harnessing Biosynthetic Networks, Protein Engineering and One‐Pot Multienzyme Cascades

Angewandte Chemie International Edition, Год журнала: 2024, Номер 63(25)

Опубликована: Апрель 15, 2024

Abstract Phenylethanoid glycosides (PhGs) exhibit a multitude of structural variations linked to diverse pharmacological activities. Assembling various PhGs via multienzyme cascades represents concise strategy over traditional synthetic methods. However, the challenge lies in identifying comprehensive set catalytic enzymes. This study explores biosynthetic PhG reconstruction from natural precursors, aiming replicate and amplify their diversity. We discovered 12 enzymes, including four novel 6′‐OH glycosyltransferases three new polyphenol oxidases, revealing intricate network biosynthesis. Subsequently, crystal structure CmGT3 (2.62 Å) was obtained, guiding identification conserved residue 144# as critical determinant for sugar donor specificity. Engineering this (FsGT61, CmGT3, FsGT6) altered recognition. Finally, one‐pot cascade established, where combined action acyltransferases boosted conversion rates by up 12.6‐fold. facilitated 26 with ranging 5–100 %, 20 additional detectable mass spectrometry. extra glycosyl hydroxyl modules demonstrated notable liver cell protection. work not only provides tools biosynthesis, but also serves proof‐of‐concept cell‐free enzymatic construction products.

Язык: Английский

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Highly Efficient Biosynthesis of Rebaudioside M9 through Enzyme Screening and Structure-Guided Engineering

Journal of Agricultural and Food Chemistry, Год журнала: 2025, Номер unknown

Опубликована: Янв. 30, 2025

Steviol glycosides (SGs) are highly valued for their sweetness, safety, and zero calories, but bitter taste low solubility limit application. Modifying glycosyl units is a promising strategy to improve sensory qualities. In this study, we identified the enzyme UGT94E13 through phylogenetic analysis screening, which catalyzes glycosylation of rebaudioside M2 (Reb M2) at C-13 position, producing novel β-1,6-O-glycosylated product M9 M9). Subsequently, catalytic activity toward Reb was enhanced 12.0-fold structure-guided engineering strategy, mechanism behind enhancement analyzed. Finally, an enzymatic cascade system comprising optimal mutant UGT94E13-F169A/I185A sucrose synthase AtSuSy constructed optimized, achieving efficient synthesis with yield 98.3% titer 42.8 g·L-1. Overall, study provides effective method enhancing SGs reference modification natural products.

Язык: Английский

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Functional characterization of a maize UDP-glucosyltransferase involved in the biosynthesis of flavonoid 7-O-glucosides and di-O-glucosides

Plant Physiology and Biochemistry, Год журнала: 2025, Номер 221, С. 109583 - 109583

Опубликована: Янв. 30, 2025

Язык: Английский

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Directed Evolution of the UDP-Glycosyltransferase UGT_BL1 for Highly Regioselective and Efficient Biosynthesis of Natural Phenolic Glycosides

Journal of Agricultural and Food Chemistry, Год журнала: 2024, Номер 72(3), С. 1640 - 1650

Опубликована: Янв. 12, 2024

The O-glycosylation of polyphenols for the synthesis glycosides has garnered substantial attention in food research applications. However, practical utility UDP-glycosyltransferases (UGTs) is significantly hindered by their low catalytic efficiency and suboptimal regioselectivity. concurrent optimization regioselectivity activity during glycosylation presents a formidable challenge. Here, we addressed long-standing activity–regioselectivity tradeoff glycosyltransferase UGTBL1 through systematic enzyme engineering. optimal combination mutants, N61S/I62M/D63W/A208R/P218W/R282W (SMWRW1W2), yielded 6.1-fold improvement relative 17.3-fold increase ratio gastrodin to para-hydroxybenzyl alcohol-4′-O-β-glucoside (with 89.5% gastrodin) compared those wild-type ultimately allowed gram-scale production (1,066.2 mg/L) using whole-cell biocatalysis. In addition, variant SMWRW1W2 exhibited preference producing phenolic from several substrates. This study lays foundation engineering additional UGTs applications regioselective retrofitting.

Язык: Английский

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Structural elucidation of the O-antigen polysaccharide from Shigatoxin-producing E. coli O179 using genetic information, NMR spectroscopy and the CASPER program

Carbohydrate Research, Год журнала: 2025, Номер 550, С. 109382 - 109382

Опубликована: Янв. 10, 2025

The serological properties of the O-antigen polysaccharide region lipopolysaccharides are used to differentiate E. coli strains into serogroups. In this study, we report structure elucidation O-specific chain O179 using NMR data, program CASPER and analysis biosynthetic information available in Database (ECODAB). presence genes that encode enzymes involved biosynthesis GDP-Man UDP-GlcA within gene cluster bacteria indicates corresponding residues could be present polysaccharide. Furthermore, occurrence four for glycosyltransferases is composed pentasaccharide repeating units; a bioinformatics approach based on predictive glycosyltransferase functions ECODAB revealed β-d-Manp-(1→4)-β-d-Manp-(1→3)-d-GlcpNAc structural element chain. spectroscopy data obtained from homonuclear heteronuclear 2D spectra (1H,1H-TOCSY, 1H,13C-HSQC, 1H,13C-H2BC 1H,13C-HMBC) were analyzed program, revealing following arrangement monosaccharide as most probable structure: →4)-α-d-GlcpA-(1→3)-[β-d-Glcp-(1→2)]β-d-Manp-(1→4)-β-d-Manp-(1→3)-β-d-GlcpNAc-(1→, which was further confirmed 1H,1H-COSY 1H,1H-NOESY spectra. α-gluconosyltransferase β-glucosyltransferase predicted alignment AlphaFold-predicted 3D structures. This shares similarities with those O6 O188, S. boydii type 16, capsular K43, explaining cross-reactivities observed belonging these O- K-antigen groups.

Язык: Английский

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Insight into the structure, oligomerization, and the role in drug resistance of human UDP-glucuronosyltransferases

Archives of Toxicology, Год журнала: 2025, Номер unknown

Опубликована: Янв. 15, 2025

Язык: Английский

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Efficient production of hydroxysalidroside in Escherichia coli via enhanced glycosylation and semi-rational design of UGT85A1

Synthetic and Systems Biotechnology, Год журнала: 2025, Номер 10(2), С. 638 - 649

Опубликована: Март 6, 2025

Hydroxysalidroside is an important natural phenylethanoid glycoside with broad application prospects in the food and pharmaceutical fields. However, its low concentration plants complex extraction hinder production. Despite being a promising way to synthesize hydroxysalidroside Escherichia coli, glycosylation remains limiting factor for A de novo biosynthetic pathway was successfully constructed E. coli via screening of glycosyltransferase, overexpressing phosphoglucomutase (pgm) UDP-glucose pyrophosphorylase (galU) ensure sufficient supply (UDPG). Additionally, semi-rational design UGT85A1 conducted expand acceptor-binding pocket eliminate steric hindrance interfering binding hydroxytyrosol. The endogenous genes ushA otsA were knocked out further reduce consumption UDPG. Finally, titer 5837.2 mg/L achieved 5 L fermenter by optimizing feeding times carbon sources. This laid foundation subsequent biosynthesis glycosides.

Язык: Английский

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Foliar application of nano Urea enhances vegetative growth of Arabidopsis thaliana over equimolar bulk urea through higher induction of biosynthesis genes but suppression of nitrogen uptake and senescence genes

Plant Growth Regulation, Год журнала: 2025, Номер unknown

Опубликована: Март 17, 2025

Язык: Английский

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