Identification and functional characterization of a new flavonoid glycosyltransferase from Rheum palmatum

doi:https://doi.org/10.1016/j.chmed.2024.08.003

Archive > Volume 17 Issue 2 > 2025,17(2):307-314. DOI:https://doi.org/10.1016/j.chmed.2024.08.003 Prev Next

掌葉大黃黃酮糖苷相關糖基轉移酶基因的鑒定及功能驗證究

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Identification and functional characterization of a new flavonoid glycosyltransferase from Rheum palmatum

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摘要:

目的：對來自大黃（Rheum palmatum）的一種糖基轉移酶（RpUGT1）進行酶學性質研究并探索其對黃酮類化合物的底物特異性。方法：利用大腸桿菌表達RpUGT1，并使用HPLC篩選其對一系列黃酮底物催化活性。質譜（MS）和核磁共振（NMR）用于確定產(chǎn)物的結構。利用同源建模、分子對接分析和定點突變研究以確定其關鍵的氨基酸殘基。結果：實驗中我們得到了重組的RpUGT1蛋白，其對多種黃酮類的化合物具有催化活性。RpUGT1能夠利用UDP葡萄糖作為糖供體，催化異鼠李素形成3-O-葡萄糖苷和山奈酚形成7-O-葡萄糖苷。為了深入了解RpUGT1的催化活性，我們進行了同源建模和分子對接分析，確定了影響其活性的關鍵殘基。定點突變研究發(fā)現(xiàn)K307在酶的催化中起到的關鍵作用。結論：RpUGT1的研究為UGT家族的作用提供了新的研究視角，并為未來研究植物中黃酮糖基化反應奠定了理論基礎。

Abstract:

Objective: To characterize a glycosyltransferase (RpUGT1) from Rheum palmatum and investigate its specificity toward flavonoid compounds. Methods: The RpUGT1 was expressed in Escherichia coli and screened for catalytic activity against a range of flavonoid substrates using a high-throughput HPLC assay method. Mass spectrometry (MS) and nuclear magnetic resonance (NMR) were used to determine the structure of the product. Homology modeling, molecular docking analyses and site-directed mutagenesis studies were conducted to identify key residues responsible for its function. Results: The recombinant RpUGT1 protein exhibited catalytic activity towards various flavonoids. Notably, RpUGT1 catalyzed the glycosylation of isorhamnetin to form 3-O-glucoside and kaempferol to form 7-O-glucoside, utilizing uridine diphosphate (UDP) glucose as the sugar donor. The homology modeling and molecular docking analyses identified key residues responsible for its activity. Subsequent sitedirected mutagenesis studies highlighted the crucial role of K307 in catalysis. Conclusion: These discoveries offer valuable perspectives on the role of the UGT family and establish a groundwork for forthcoming research on the synthesis of flavonoids in plants.

關鍵詞:

黃酮;糖基轉移酶;同源建模;分子對接;掌葉大黃;定點突變

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This work was financially supported by National Natural Science Foundation (No. 82204567, 8220142901, 82104334), Hebei Natural Science Foundation (No. H2023201005), Shaanxi Provincial Key Industry Innovation Chain (Group) Project (No. 2021ZDLSF04-01), China University Student Innovation and Entrepreneurship Training Program Project (No. 202310075016).

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