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Updated: Jul 10, 2026

Unraveling Entropic Rate Acceleration Induced by Solvent Dynamics in Membrane Enzymes
Published on: January 16, 2016
Engineering AaADH1 for enhanced catalytic efficiency and solubility via substrate tunnel modulation
Chenyu Wang1, Xiao Feng2, Lixia Zong3
1NHC Key Laboratory of Biotechnology of Antibiotics, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China; School of Pharmacy, North China University of Science and Technology, Tangshan, Hebei 063210, China.
Abstract:
Artemisinic alcohol dehydrogenase 1 (AaADH1) plays a pivotal role in the biosynthesis of artemisinic acid, a key precursor of the antimalarial drug artemisinin. However, its low catalytic activity and poor yield limit its industrial application. In this study, we employed a structure-guided protein engineering approach to improve the enzymatic properties of AaADH1. Substrate tunnel analysis identified key bottleneck residues that were subjected to alanine scanning mutagenesis. Among the resulting mutants, L366A displayed a 30% increase in activity and a 53% enhancement in catalytic efficiency (kcat/KM), along with a 5-fold higher expression level than the wild type (WT). Consequently, a saturation mutagenesis was performed at position 366. Compared to the WT, the kcat/KM of the L366V, L366I, and L366F mutants increased by 100%, 38%, and 16%, respectively. Molecular dynamics simulations revealed that mutations at L366 enhanced the flexibility of the catalytic pocket and stabilized the local secondary structure, thereby contributing to both improved activity and expression. These findings provide a rational framework for further optimization of AaADH1 for industrial artemisinic acid production.
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