Steviol glycosides are ideal natural sweeteners, among which rebaudioside D (Reb D) is widely favored due to its high sweetness and low bitterness.Currently, it is mainly synthesized enzymatically from rebaudioside A (Reb A) by glycosyltransferases such as YojK, UGTSL2, and PgUGT.However, systematic studies on these enzymes remain limited.In this study, bioinformatics analysis combined with molecular simulations was used to comprehensively investigate the sequence and structural features of glycosyltransferases involved in Reb D biosynthesis, and to elucidate the molecular mechanisms underlying improved enzymatic performance.Results revealed that the active site regions exhibited higher structural conservation than the overall proteins, with highly conserved hydrophobic cyclic side-chain residues.Moreover, improved catalytic efficiency was not dependent on increased stability or hydrogen bond quantity, but was more closely related to the distance between catalytic atoms and the synergistic positioning of substrates.Energy and dynamic hydrogen bond network analyses indicated that binding between enzymes and Reb A was mainly driven by hydrophobic interactions.The hydrogen bonding interactions at both glycosyl ends were significantly stronger than those at the aglycone moiety, and the mutations further enhanced these hydrogen bond interactions.Based on these findings, a substrate binding mechanism model was proposed, providing a theoretical basis for the efficient enzymatic conversion of Reb D.
WU Xinming
,
ZHENG Yue
,
LIU Fufeng
. Mechanistic insights into rebaudioside D-producing glycosyltransferases via molecular simulations[J]. Food and Fermentation Industries, 2026
, 52(8)
: 9
-20
.
DOI: 10.13995/j.cnki.11-1802/ts.044006
[1] MORISSETTE A, DE WOUTERS D’OPLINTER A, ANDRE D M, et al.Rebaudioside D decreases adiposity and hepatic lipid accumulation in a mouse model of obesity[J].Scientific Reports, 2024, 14(1):3077.
[2] SONG F W, WANG S D, MAO Y H, et al.Enhancing rebaudioside M synthesis via introducing sulfur-mediated interactions between glycosyltransferase UGT76G1 and rebaudioside D[J].Journal of Agricultural and Food Chemistry, 2025, 73(1):667-677.
[3] GUO B D, HOU X D, ZHANG Y, et al.Highly efficient production of rebaudioside D enabled by structure-guided engineering of bacterial glycosyltransferase YojK[J].Frontiers in Bioengineering and Biotechnology, 2022, 10:985826.
[4] ZHANG S S, YANG Y S, LYU C C, et al.Identification of the key residues of the uridine diphosphate glycosyltransferase 91D2 and its effect on the accumulation of steviol glycosides in Stevia rebaudiana[J].Journal of Agricultural and Food Chemistry, 2021, 69(6):1852-1863.
[5] CHEN L L, CAI R X, WENG J Y, et al.Production of rebaudioside D from stevioside using a UGTSL2 Asn358Phe mutant in a multi-enzyme system[J].Microbial Biotechnology, 2020, 13(4):974-983.
[6] MA S Y, MA Y Y.A sustainable strategy for biosynthesis of Rebaudioside D using a novel glycosyltransferase of Solanum tuberosum[J].Biotechnology Journal, 2024, 19(2):e2300628.
[7] CHEN M Q, SONG F W, QIN Y X, et al.Improving thermostability and catalytic activity of glycosyltransferase from Panax ginseng by semi-rational design for rebaudioside D synthesis[J].Frontiers in Bioengineering and Biotechnology, 2022, 10:884898.
[8] TALEVI A.Computer-aided drug discovery and design:Recent advances and future prospects[J].Methods in Molecular Biology 2024, 2714:1-20.
[9] YAGI H, TAKAGI K, KATO K.Exploring domain architectures of human glycosyltransferases:Highlighting the functional diversity of non-catalytic add-on domains[J].Biochimica et Biophysica Acta-General Subjects, 2024, 1868(10):130687.
[10] XIA H, ZHANG Z H, DING J, et al.A glycosyltransferase from Arabidopsis thaliana enables the efficient enzymatic synthesis of gastrodin[J].Catalysis Letters, 2024, 154(4):1558-1566.
[11] YANG M L, YANG S, DENG Z W, et al.Improving the thermostability of glycosyltransferase UGT76G1 by computer-aided target analysis for highly efficient biosynthesis of rebaudioside M[J].Food Bioscience, 2023, 56:103119.
[12] SARAIVA A, CARRASCOSA C, RAMOS F, et al.Agave syrup:Chemical analysis and nutritional profile, applications in the food industry and health impacts[J].International Journal of Environmental Research and Public Health, 2022, 19(12):7022.
[13] CHEN Y X, XIE Y N, AJUWON K M, et al.Xylo-oligosaccharides, preparation and application to human and animal health:A review[J].Frontiers in Nutrition, 2021, 8:731930.
[14] SIDDIQUI H, SAMI F, HAYAT S.Glucose:Sweet or bitter effects in plants-a review on current and future perspective[J].Carbohydrate Research, 2020, 487:107884.
[15] FORLI S, HUEY R, PIQUE M E, et al.Computational protein-ligand docking and virtual drug screening with the AutoDock suite[J].Nature Protocols, 2016, 11(5):905-919.
[16] ASSAREH E, MEHRNEJAD F, ASGHARI S M.Structural studies on an anti-angiogenic peptide using molecular modeling[J].Iranian Journal of Biotechnology, 2020, 18(4):e2553.
[17] HUANG W, LIN Z X, VAN GUNSTEREN W F.Validation of the GROMOS 54A7 force field with respect to β-peptide folding[J].Journal of Chemical Theory and Computation, 2011, 7(5):1237-1243.
[18] SARGSYAN K, GRAUFFEL C, LIM C.How molecular size impacts RMSD applications in molecular dynamics simulations[J].Journal of Chemical Theory and Computation, 2017, 13(4):1518-1524.
[19] CHOI J M, VUPPALA S, PARK M J, et al.Computer simulation approach to the identification of visfatin-derived angiogenic peptides[J].PLoS One, 2023, 18(6):e0287577.
[20] WANG B, KENNEDY M A.Principal components analysis of protein sequence clusters[J].Journal of Structural and Functional Genomics, 2014, 15(1):1-11.
[21] AHARONOVSKY E, TRIFONOV E N.Protein sequence modules[J].Journal of Biomolecular Structure and Dynamics, 2005, 23(3):237-242.
[22] CAMPITELLI P, MODI T, KUMAR S, et al.The role of conformational dynamics and allostery in modulating protein evolution[J].Annual Review of Biophysics, 2020, 49:267-288.
[23] KARNATI K R, WANG Y X.Structural and binding insights into HIV-1 protease and P2-ligand interactions through molecular dynamics simulations, binding free energy and principal component analysis[J].Journal of Molecular Graphics and Modelling, 2019, 92:112-122.
[24] MAGUIRE J B, BOYKEN S E, BAKER D, et al.Rapid sampling of hydrogen bond networks for computational protein design[J].Journal of Chemical Theory and Computation, 2018, 14(5):2751-2760.
[25] LIN M, WANG F, ZHU Y S.Modeled structure-based computational redesign of a glycosyltransferase for the synthesis of rebaudioside D from rebaudioside A[J].Biochemical Engineering Journal, 2020, 159:107626.
