Guo Zhihan, Li Pengfei, Huang Chaokang, Niu Tengfei, Wang Ziyan, Lai Guanxue, Ding Lili, Yang Li, Wang Zhengtao, Pu Zhongji, Wang Rufeng
Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China; The MOE Key Laboratory for Standardization of Chinese Medicines, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China.
Hefei MiQro Era Digital Technology Co. Ltd., Hefei, China.
Int J Biol Macromol. 2025 May;308(Pt 2):142539. doi: 10.1016/j.ijbiomac.2025.142539. Epub 2025 Mar 25.
The limited abundance of xylosylated ginsenosides and the lack of efficient biocatalysts hinder their pharmacological exploration. This study identified a 2"-O-xylosyltransferase (PnUGT57) from Panax notoginseng that catalyzes the conversion of ginsenoside Rg3 to notoginsenoside ST4. Wild-type PnUGT57 preferred UDP-xylose over UDP-glucose and UDP-rhamnose and displayed limited thermostability (t = 6.73 h at 30 °C). To enhance UDP-xylose specificity, sequence-guided mutagenesis generated the C140A variant, which achieved remarkable UDP-xylose specificity (100 % conversion) with a 1.34-fold increase in catalytic efficiency while showing weak activity toward UDP-glucose (8.7 %) and UDP-rhamnose (5.2 %) activity. The F367A mutant possesses only xylosyltransferase activity but with reduced catalytic efficiency (0.3-fold of the WT). Molecular docking revealed that the enhanced UDP-xylose specificity in C140A and F367A resulted from the loss of key hydrogen bonding and hydrophobic interactions. To improve thermostability, computational design produced a triple mutant (P101S/L200C/G255D) with an 8.58-fold longer half-life (57.76 h), attributed to optimized surface charge distribution and improved hydration layer formation, as confirmed by molecular dynamics simulation. The combinatorial mutant C140A/P101S/L200C/G255D synergistically improved UDP-xylose specificity, thermostability, and catalytic efficiency, enabling efficient ST4 biosynthesis. This study elucidates the catalytic mechanism of PnUGT57 and presents engineered variants as promising biocatalysts for sustainable ginsenoside production.
木糖基化人参皂苷的有限丰度以及缺乏高效的生物催化剂阻碍了它们的药理学研究。本研究从三七中鉴定出一种2″-O-木糖基转移酶（PnUGT57），该酶催化人参皂苷Rg3转化为三七皂苷ST4。野生型PnUGT57对UDP-木糖的偏好高于UDP-葡萄糖和UDP-鼠李糖，并且热稳定性有限（30℃下t = 6.73小时）。为了提高UDP-木糖特异性，通过序列引导诱变产生了C140A变体，该变体实现了显著的UDP-木糖特异性（100%转化），催化效率提高了1.34倍，同时对UDP-葡萄糖（8.7%）和UDP-鼠李糖（活性5.2%）显示出较弱的活性。F367A突变体仅具有木糖基转移酶活性，但催化效率降低（为野生型的0.3倍）。分子对接表明，C140A和F367A中UDP-木糖特异性的增强是由于关键氢键和疏水相互作用的丧失。为了提高热稳定性，通过计算设计产生了一个半衰期延长8.58倍（57.76小时）的三重突变体（P101S/L200C/G255D），这归因于优化的表面电荷分布和改善的水合层形成，分子动力学模拟证实了这一点。组合突变体C140A/P101S/L200C/G255D协同提高了UDP-木糖特异性、热稳定性和催化效率，实现了ST4的高效生物合成。本研究阐明了PnUGT57的催化机制，并展示了工程变体作为可持续人参皂苷生产的有前景的生物催化剂。
