Ngawiset Sirikul, Ismail Abbas, Murakami Shuichiro, Pongsawasdi Piamsook, Rungrotmongkol Thanyada, Krusong Kuakarun
Center of Excellence in Structural and Computational Biology, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand.
Department of Agricultural Chemistry, School of Agriculture, Meiji University, Kawasaki-shi, Kanagawa 214-8571, Japan.
Comput Struct Biotechnol J. 2023 Jan 10;21:899-909. doi: 10.1016/j.csbj.2023.01.011. eCollection 2023.
Amylomaltase can be used to synthesize large ring cyclodextrins (LR-CDs), applied as drug solubilizer, gene delivery vehicle and protein aggregation suppressor. This study aims to determine the functional amino acid positions of amylomaltase (AM) involved in LR-CD synthesis by site-directed mutagenesis approach and molecular dynamic simulation. Mutants named Δ167, Y23A, P228Y, E231Y, A413F and G417F were constructed, purified, and characterized. The truncated AM, Δ167 exhibited no starch transglycosylation activity, indicating that the N-terminal domain of AM is necessary for enzyme activity. The P228Y, A413F and G417F produced larger LR-CDs from CD36-CD40 as compared to CD29 by WT. A413F and G417F mutants produced significantly low LR-CD yield compared to the WT. The A413F mutation affected all tested enzyme activities (starch tranglycosylation, disproportionation and cyclization), while the G417F mutation hindered the cyclization activity. P228Y mutation significantly lowered the of disproportionation activity, while E231Y mutant exhibited much higher and values for starch transglycosylation, compared to that of the WT. In addition, Y23A mutation affected the kinetic parameters of starch transglycosylation and cyclization. Molecular dynamic simulation further confirmed these mutations' impacts on the AM and LR-CD interactions. Identified functional amino acids for LR-CD synthesis may serve as a model for future modification to improve the properties and yield of LR-CDs.
淀粉麦芽糖酶可用于合成大环环糊精（LR - CD），用作药物增溶剂、基因传递载体和蛋白质聚集抑制剂。本研究旨在通过定点诱变方法和分子动力学模拟确定参与LR - CD合成的淀粉麦芽糖酶（AM）的功能氨基酸位置。构建、纯化并表征了名为Δ167、Y23A、P228Y、E231Y、A413F和G417F的突变体。截短的AM，即Δ167，没有淀粉转糖基化活性，这表明AM的N端结构域对酶活性是必需的。与野生型（WT）相比，P228Y、A413F和G417F从CD36 - CD40产生的LR - CD比从CD29产生的更大。与WT相比，A413F和G417F突变体产生的LR - CD产量显著较低。A413F突变影响了所有测试的酶活性（淀粉转糖基化、歧化和环化），而G417F突变阻碍了环化活性。P228Y突变显著降低了歧化活性的 ，而与WT相比，E231Y突变体在淀粉转糖基化方面表现出更高的 和 值。此外，Y23A突变影响了淀粉转糖基化和环化的动力学参数。分子动力学模拟进一步证实了这些突变对AM与LR - CD相互作用的影响。确定的参与LR - CD合成的功能氨基酸可作为未来修饰的模型，以改善LR - CD的性质和产量。
