Glycosyltransferases: Mining, engineering and applications in biosynthesis of glycosylated plant natural products

Synthetic and Systems Biotechnology, Год журнала: 2022, Номер 7(1), С. 602 - 620

Опубликована: Фев. 2, 2022

UDP-Glycosyltransferases (UGTs) catalyze the transfer of nucleotide-activated sugars to specific acceptors, among which GT1 family enzymes are well-known for their function in biosynthesis natural product glycosides. Elucidating GT represents necessary step metabolic engineering aglycone glycosylation produce drug leads, cosmetics, nutrients and sweeteners. In this review, we systematically summarize phylogenetic distribution catalytic diversity plant GTs. We also discuss recent progress identification novel candidates synthesis products (PNPs) using multi-omics technology deep learning predicted models. highlight advances rational design directed evolution strategies new or improved functions. Finally, cover breakthroughs application GTs microbial some representative glycosylated PNPs, including flavonoid glycosides (fisetin 3-

Язык: Английский

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Structure-function and engineering of plant UDP-glycosyltransferase

Computational and Structural Biotechnology Journal, Год журнала: 2023, Номер 21, С. 5358 - 5371

Опубликована: Янв. 1, 2023

Natural products synthesized by plants have substantial industrial and medicinal values are therefore attracting increasing interest in various related industries. Among the key enzyme families involved biosynthesis of natural products, uridine diphosphate-dependent glycosyltransferases (UGTs) play a crucial role plants. In recent years, significant efforts been made to elucidate catalytic mechanisms substrate recognition plant UGTs improve them for desired functions. this review, we presented comprehensive overview all currently published structures UGTs, along with in-depth analyses corresponding mechanisms. addition, summarized evaluated protein engineering strategies applied activities particular focus on high-throughput screening methods. The primary objective review is provide readers understanding serve as valuable reference latest techniques used their activities.

Язык: Английский

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Insights into the missing apiosylation step in flavonoid apiosides biosynthesis of Leguminosae plants

Nature Communications, Год журнала: 2023, Номер 14(1)

Опубликована: Окт. 20, 2023

Apiose is a natural pentose containing an unusual branched-chain structure. Apiosides are bioactive products widely present in the plant kingdom. However, little known on key apiosylation reaction biosynthetic pathways of apiosides. In this work, we discover apiosyltransferase GuApiGT from Glycyrrhiza uralensis. could efficiently catalyze 2″-O-apiosylation flavonoid glycosides, and exhibits strict selectivity towards UDP-apiose. We further solve crystal structure GuApiGT, determine sugar-binding motif (RLGSDH) through structural analysis theoretical calculations, obtain mutants with altered sugar protein engineering. Moreover, 121 candidate genes Leguminosae plants, identify functions 4 enzymes. Finally, introduce its upstream into Nicotiana benthamiana, complete de novo biosynthesis series This work reports efficient phenolic apiosyltransferase, reveals mechanisms for donor selectivity.

Язык: Английский

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Sustainable bioproduction of natural sugar substitutes: Strategies and challenges

Trends in Food Science & Technology, Год журнала: 2022, Номер 129, С. 512 - 527

Опубликована: Ноя. 1, 2022

Язык: Английский

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Functional characterization, structural basis, and protein engineering of a rare flavonoid 2′-O-glycosyltransferase from Scutellaria baicalensis

Acta Pharmaceutica Sinica B, Год журнала: 2024, Номер 14(8), С. 3746 - 3759

Опубликована: Апрель 5, 2024

Glycosylation is an important post-modification reaction in plant secondary metabolism, and contributes to structural diversity of bioactive natural products. In plants, glycosylation usually catalyzed by UDP-glycosyltransferases. Flavonoid 2'-O-glycosides are rare glycosides. However, no UGTs have been reported, thus far, specifically catalyze 2'-O-glycosylation flavonoids. this work, UGT71AP2 was identified from the medicinal Scutellaria baicalensis as first flavonoid 2'-O-glycosyltransferase. It could preferentially transfer a glycosyl moiety 2'-hydroxy at least nine flavonoids yield six new compounds. Some showed noticeable inhibitory activities against cyclooxygenase 2. The crystal structure (2.15 Å) solved, mechanisms its regio-selectivity interpreted pK calculations, molecular docking, MD simulation, MM/GBSA binding free energy, QM/MM, hydrogen‒deuterium exchange mass spectrometry analysis. Through structure-guided rational design, we obtained L138T/V179D/M180T mutant with remarkably enhanced (the ratio 7-O-glycosylation byproducts decreased 48% 4%) catalytic efficiency (k cat/K m, 0.23 L/(s·μmol), 12-fold higher than native). Moreover, also possesses moderate UDP-dependent de-glycosylation activity, dual function glycosyltransferase. This work provides efficient biocatalyst sets good example for protein engineering optimize enzyme features through design.

Язык: Английский

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Functional and structural dissection of glycosyltransferases underlying the glycodiversity of wolfberry-derived bioactive ingredients lycibarbarspermidines

Nature Communications, Год журнала: 2024, Номер 15(1)

Опубликована: Май 30, 2024

Abstract Lycibarbarspermidines are unusual phenolamide glycosides characterized by a dicaffeoylspermidine core with multiple glycosyl substitutions, and serve as major class of bioactive ingredients in the wolfberry. So far, little is known about enzymatic basis glycosylation phenolamides including dicaffeoylspermidine. Here, we identify five lycibarbarspermidine glycosyltransferases, LbUGT1-5, which first phenolamide-type glycosyltransferases catalyze regioselective dicaffeoylspermidines to form structurally diverse lycibarbarspermidines Notably, LbUGT3 acts distinctive enzyme that catalyzes tandem sugar transfer ortho-dihydroxy group on caffeoyl moiety ortho-diglucosylated product, while LbUGT1 accurately discriminates dihydrocaffeoyl groups site-selective transfer. Crystal structure analysis complexes UDP, combined molecular dynamics simulations, revealed structural difference selectivity between LbUGT3. Site-directed mutagenesis illuminates conserved tyrosine residue (Y389 Y390 LbUGT3) PSPG box plays crucial role regulating regioselectivity Our study thus sheds light underpinnings chemical diversity wolfberry, expands repertoire nature.

Язык: Английский

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Structural insights into the catalytic selectivity of glycosyltransferase SgUGT94-289-3 towards mogrosides

Nature Communications, Год журнала: 2024, Номер 15(1)

Опубликована: Июль 30, 2024

Mogrosides constitute a series of natural sweeteners extracted from Siraitia grosvenorii fruits. These mogrosides are glucosylated to different degrees, with mogroside V (M5) and siamenoside I (SIA) being two high intensities sweetness. SgUGT94-289-3 constitutes uridine diphosphate (UDP)-dependent glycosyltransferase (UGT) responsible for the biosynthesis M5 SIA, by continuously catalyzing glucosylation on IIe (M2E) subsequent intermediate products. However, mechanism its promiscuous substrate recognition multiple catalytic modes remains unclear. Here, we report complex structures enzymatic characterization SgUGT94-289-3. We show that adopts dual-pocket organization in active site, which allows structurally distinct reactive ends be presented pockets site reaction, thus enabling both promiscuity regioselectivity. further identified structural motif is essential activity regioselectivity, generated mutants greatly improved M5/SIA production M2E an vitro one-pot setup. authors present mechanistic characterisation UDP-dependent SgUGT94-289-3, generates interest as sweeteners. Structure-based engineering yielded variants specificity activity.

Язык: Английский

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Functional Characterization and Protein Engineering of a Triterpene 3‐/6‐/2′‐O‐Glycosyltransferase Reveal a Conserved Residue Critical for the Regiospecificity

Angewandte Chemie International Edition, Год журнала: 2021, Номер 61(8)

Опубликована: Дек. 11, 2021

Engineering the function of triterpene glucosyltransferases (GTs) is challenging due to large size sugar acceptors. In this work, we identified a multifunctional glycosyltransferase AmGT8 catalyzing 3-/6-/2'-O-glycosylation from medicinal plant Astragalus membranaceus. To engineer its regiospecificity, small mutant library was built based on semi-rational design. Variants A394F, A394D, and T131V were found catalyze specific 6-O, 3-O, 2'-O glycosylation, respectively. The origin regioselectivity A394F variant studied by molecular dynamics hydrogen deuterium exchange mass spectrometry. Residue 394 highly conserved as A/G critical for regiospecificity C- O-GTs TcCGT1 GuGT10/14. Finally, astragalosides III IV synthesized mutants P192E. This work reports biocatalysts saponin synthesis gives new insights into protein engineering in GTs.

Язык: Английский

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Molecular characterization and structural basis of a promiscuous glycosyltransferase for β‐(1,6) oligoglucoside chain glycosides biosynthesis

Plant Biotechnology Journal, Год журнала: 2025, Номер unknown

Опубликована: Март 19, 2025

Summary Sugar building blocks are crucial for the chemical diversity and biological activity of secondary metabolites. UDP‐dependent glycosyltransferases (UGTs) play a pivotal role in biosynthesis glycosides plants by catalysing attachment sugar moieties to various bioactive natural products. However, oligosaccharide‐chain is often limited narrow substrate specificity UGTs. In this study, we identify regio‐specific β ‐(1,6) glycosyltransferase, UGT94BY1, from Platycodon grandiflorum . UGT94BY1 exhibits broad promiscuity can transfer up three C6‐OH position glucosyl group triterpenoids phenolic glycosides, thereby forming oligoglucoside chains. To elucidate mechanism underlying its selectivity, determined crystal structure complex with UDP at resolution 2.0 Å. Molecular simulations revealed that critical structural motif, comprising residues N84‐M91, S141‐L155 R179‐E186, plays key recognizing acceptors facilitating chain elongation. Our study unveils powerful glycosyltransferase highlights regions involved recognition extension, providing valuable insights designing UGTs customized specificities biotechnological applications.

Язык: Английский

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β-Carotene alleviates substrate inhibition caused by asymmetric cooperativity

Nature Communications, Год журнала: 2025, Номер 16(1)

Опубликована: Март 29, 2025

Enzymes are essential catalysts in biological systems. Substrate inhibition, once dismissed, is now observed 20% of enzymes1 and attributed to the formation an unproductive enzyme-substrate complex, with no structural evidence unproductivity provided date1-6. This study uncovers molecular mechanism substrate inhibition tobacco glucosyltransferase NbUGT72AY1, which transfers glucose phenols for plant protection. The peculiarity that β-carotene strongly attenuates despite being a competitive inhibitor, allows determine conformational changes occur during binding both active substrate-inhibited complexes. Crystallography reveals structurally different ternary complexes do not conform classical mechanisms. An alternative pathway suggests substrates bind randomly, but reaction occurs only if specific order followed (asymmetric cooperativity). unreported paradigm explains reactivation by inhibitors, opening new research avenues metabolic regulation industrial applications.

Язык: Английский

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