State Key Laboratory of Southwestern Chinese Medicine Resources, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China.
State Key Laboratory for Quality Ensurance and Sustainable Use of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, P. R. China.
J Agric Food Chem. 2024 Aug 14;72(32):18214-18224. doi: 10.1021/acs.jafc.4c04235. Epub 2024 Aug 5.
Mogrosides are natural compounds highly valued in the food sector for their exceptional sweetness. Here, we report a novel -glycosyltransferase (UGT74DD1) from that catalyzes the conversion of mogrol to mogroside IIE. Site-directed mutagenesis yielded the UGT74DD1-W351A mutant, which exhibited the new capability to transform mogroside IIE into the valuable sweetener mogroside III, but with low catalytic activity. Subsequently, using structure-guided directed evolution with combinatorial active-site saturation testing, the superior mutant M6 (W351A/Q373 K/E49H/Q335W/S278C/D17F) were obtained, which showed a 46.1-fold increase in catalytic activity compared to UGT74DD1-W351A. Molecular dynamics simulations suggested that the enhanced activity and extended substrate profiles of M6 are due to its enlarged substrate-binding pocket and strengthened enzyme-substrate hydrogen bonding interactions. Overall, we redesigned UGT74DD1, yielding mutants that catalyze the conversion of mogrol into mogroside III. This study thus broadens the toolbox of UGTs capable of catalyzing the formation of valuable polyglycoside compounds.
罗汉果苷是一种天然化合物，因其极高的甜度而在食品领域受到高度重视。在这里，我们报道了一种新型的 -糖基转移酶（UGT74DD1），它能催化 mogrol 转化为 mogroside IIE。定点突变得到了 UGT74DD1-W351A 突变体，该突变体具有将 mogroside IIE 转化为有价值的甜味剂 mogroside III 的新能力，但催化活性较低。随后，我们通过基于结构的定向进化和组合活性位点饱和测试，得到了具有优越催化活性的突变体 M6（W351A/Q373 K/E49H/Q335W/S278C/D17F），与 UGT74DD1-W351A 相比，其催化活性提高了 46.1 倍。分子动力学模拟表明，M6 增强的活性和扩展的底物谱是由于其扩大的底物结合口袋和增强的酶-底物氢键相互作用。总之，我们重新设计了 UGT74DD1，得到了能够催化 mogrol 转化为 mogroside III 的突变体。该研究拓宽了能够催化形成有价值的多糖化合物的 UGT 工具包。
