疟疾是主要由恶性疟原虫和间日疟原虫引起的严重传染病，2020年，全球报告了约2.41亿疟疾病例，其中约有627,000人死于该疾病，五岁以下的儿童占疟疾死亡人数的77%。青蒿素联合疗法(ACTs)是世界卫生组织推荐的最有效的临床治疗疟疾感染的方法，然而传统的植物提取易受季节、地域的影响导致青蒿素产量不稳定，无法满足商业化的大量需求。合成生物学的发展为天然药物的生物合成提供了新的思路，Keasling团队在酵母细胞中设计并整合了青蒿酸的生物合成途径，青蒿酸产量可达25 g L^–1，再经过化学半合成最终获得青蒿素。对青蒿酸生物合成途径中的关键合成元件进行探索，发现影响青蒿酸最终合成的关键步骤在于最后两步的氧化还原反应，其中导入青蒿醇脱氢酶(AaADH1)能够在原有酵母工程菌的基础上提高80%的青蒿酸产量，表明AaADH1在青蒿酸的生物合成中发挥重要作用，本文对AaADH1开展了蛋白结构和催化机制研究。

       本研究通过大肠杆菌(E. coli)表达系统对AaADH1进行异源表达，经过Co²⁺-NTA亲和层析和强阴离子交换色谱法纯化，得到纯度在90%以上的可溶性蛋白，运用GC-MS法测定蛋白活性。采用悬滴法对AaADH1本体和AaADH1-NAD⁺复合物进行蛋白结晶的初筛和结晶条件优化，获得了分辨率分别为2.95 Å和1.80 Å的衍射数据，成功解析了AaADH1本体(PDB: 7CYI)及AaADH1-NAD⁺复合物(PDB: 6LJH)的三维结构，对整体折叠、锌的配位以及潜在的底物结合位点进行了详细的结构分析。以AaADH1-NAD⁺复合物结构为基础，将青蒿醇(ART)对接到AaADH1-NAD⁺结构的活性口袋，构建AaADH1-NAD⁺-ART三元复合物结构并进行分子动力学模拟优化，从而选取稳定构象进行QM/MM MD (Quantum Mechanical/Molecular Mechanical Molecular Dynamics)模拟其脱氢反应机理，重点对锌的配位情况以及负氢和质子转移的详尽过程进行研究，并通过比较活化自由能的计算值与实验值和关键氨基酸突变体的活性验证推测机制的合理性。结果表明底物醇与锌的配位不是机理中的必需步骤，负氢和质子的转移以“准协同”方式进行，这是由青蒿醇独特的烯丙醇式化学结构决定的，丰富了现有的醇脱氢酶催化反应的分子机制。本文还对AaADH1中底物结合腔和底物通道的关键氨基酸进行半理性设计，采用点饱和突变技术构建“小而精”的突变体文库，配合CCK8法筛选得到活性和可溶性均有所提高的突变体L366A。

       综上所述，本研究成功解析了AaADH1本体和AaADH1-NAD⁺二元复合物的蛋白三维结构，通过量化计算和分子动力学模拟等阐述了AaADH1催化青蒿醇生成青蒿醛的分子机制。值得注意的是，AaADH1遵循一种非常规且特定的催化机理，其中质子和负氢转移以“准协同”的方式进行。本研究拓宽了对醇脱氢酶催化机制的认识，进一步理解了青蒿素的生物合成途径，还为合理设计青蒿素生物合成途径以提高青蒿素的产量来满足疟疾患者的供应需求提供了启示。

Malaria is a serious infectious disease mainly caused by Plasmodium falciparum and Plasmodium vivax. In 2020, approximately 241 million malaria cases were reported globally, among which about 627,000 died from that disease, with children under the age of 5 accounting for 77% of all malaria deaths. Artemisinin-combination therapies (ACTs) are the most effective clinical treatment methods recommended by the World Health Organization (WHO). However, the plant extraction of artemisinin could be affected by seasons and regions, resulting in unstable yields, which cannot meet its commercial needs. The development of synthetic biology has provided new ideas for the biosynthesis of natural drugs. Keasling’s group designed and integrated the biosynthetic pathway of artemisinic acid in engineered yeast cells. The production of artemisinic acid in Saccharomyces cerevisiae can reach 25 g L^–1, and finally obtain artemisinin using a chemical semi-synthesis method. The key synthesis elements in the biosynthetic pathway of artemisinic acid were explored, and it was found that the key steps affecting the final synthesis of artemisinic acid are the redox reactions of the last two steps, in which the incorporation of Artemisia annua alcohol dehydrogenase 1 (AaADH1) gene into the engineered yeast strain led to an 80% increase of artemisinic acid production, indicating that AaADH1 plays an important role in the biosynthesis of artemisinic acid. In this paper, the protein structures and catalytic mechanism of AaADH1 were studied.

 In this study, AaADH1 was heterologously expressed by E. coli expression system, purified by Co²⁺-NTA affinity chromatography and anion exchange chromatography, and the soluble protein with a purity of more than 90% was obtained. The GC-MS method was used to detect the protein activity. The hanging-drop method was used for crystallization of apo AaADH1 and AaADH1-NAD⁺ complex. After the primary screening and crystallization conditions optimization, diffraction data of the crystals were obtained at resolutions of 2.95 Å and 1.80 Å, respectively. The three-dimensional structures of apo AaADH1 (PDB: 7CYI) and AaADH1-NAD⁺ complex (PDB: 6LJH) were successfully resolved, and detailed structural analysis of the overall folding, coordination of zinc, and potential substrate binding sites was performed. Based on the structure of AaADH1-NAD⁺ complex, artemisinic alcohol (ART) was docked into the active pocket of the binary structure, and the AaADH1-NAD⁺-ART ternary complex structure was constructed and optimized by molecular dynamics simulations. The stable conformation was selected for QM/MM MD (Quantum Mechanical/Molecular Mechanical Molecular Dynamics) to simulate its dehydrogenation reaction mechanism, focusing on the study of the coordination of zinc and the detailed process of hydride and proton transfer, and rationality of the inferred mechanism was verified the by comparing the calculated and experimental values of activation free energy and determination of the activities of key amino acid mutants. The results show that the coordination of substrate alcohols with zinc is not a necessary step in the mechanism, and the transfer of hydride and proton proceed in a "quasi-concerted" manner, which is determined by the unique allyl alcohol-like chemical structure of ART, which enriches the present molecular mechanisms of the reactions catalyzed by alcohol dehydrogenases. In this paper, the semi-rational design of key amino acids in the substrate binding cavity and substrate channel of AaADH1 was carried out, and a "small and refined" mutant library was constructed by using point saturation mutation technology. The mutant L366A with improved activity and solubility was screened with CCK8 method.

In conclusion, this study successfully analyzed the three-dimensional protein structures of apo AaADH1 and AaADH1-NAD⁺ binary complex, and explained the molecular mechanism of AaADH1 catalyzing ART to artemisinic aldehyde by QM/MM MD simulations. Notably, AaADH1 follows an unconventional and specific catalytic mechanism in which proton and hydride transfer proceed in a "quasi-concerted" manner. This study broadens the understanding of the catalytic mechanisms of alcohol dehydrogenases, further understands the biosynthetic pathway of artemisinin, and also sheds light on the rational design of artemisinin biosynthetic pathways to increase the production of artemisinin to meet the supply needs of malaria patients.