Abstract:
D-amino acid oxidase (DAAO)-catalyzed selective oxidative deamination is a very promising process for synthesizing l-amino acids including l-phosphinothricin (l-PPT, a high-efficiency and broad-spectrum herbicide). However, the wild-type DAAO\'s low activity toward unnatural substrates like d-phosphinothricin (d-PPT) hampers its application. Herein, a DAAO from Caenorhabditis elegans (CeDAAO) was screened and engineered to improve the catalytic potential on d-PPT. First, we designed a novel growth selection system, taking into account the intricate relationship between the growth of Escherichia coli (E. coli) and the catalytic mechanism of DAAO. The developed system was used for high-throughput screening of gene libraries, resulting in the discovery of a variant (M6) with significantly increased catalytic activity against d-PPT. The variant displays different catalytic properties on substrates with varying hydrophobicity and hydrophilicity. Analysis using Alphafold2 modeling and molecular dynamic simulations showed that the reason for the enhanced activity was the substrate-binding pocket with enlarged size and suitable charge distribution. Further QM/MM calculations revealed that the crucial factor for enhancing activity lies in reducing the initial energy barrier of the reductive half reaction. Finally, a comprehensive binding-model index to predict the enhanced activity of DAAO toward d-PPT, and an enzymatic deracemization approach was developed, enabling the efficient synthesis of l-PPT with remarkable efficiency.
摘要:
D-氨基酸氧化酶(DAAO)催化的选择性氧化脱氨基是合成包括l-膦丝菌素在内的l-氨基酸的非常有前途的方法(l-PPT,高效广谱除草剂)。然而,野生型DAAO对非天然底物如d-膦丝菌素(d-PPT)的低活性阻碍了其应用。在这里,对来自秀丽隐杆线虫(CeDAAO)的DAAO进行筛选和改造,以提高d-PPT的催化潜力。首先,我们设计了一个新颖的生长选择系统,考虑到大肠杆菌生长之间的复杂关系(E.大肠杆菌)和DAAO的催化机理。所开发的系统用于基因文库的高通量筛选,导致发现具有显著增加的针对d-PPT的催化活性的变体(M6)。该变体在具有不同疏水性和亲水性的底物上显示不同的催化性质。使用Alphafold2建模和分子动力学模拟的分析表明,活性增强的原因是底物结合袋具有更大的尺寸和合适的电荷分布。进一步的QM/MM计算表明,增强活性的关键因素在于降低还原半反应的初始能垒。最后,一个综合的结合模型指数来预测DAAO对d-PPT的活性增强,并开发了一种酶促除菌方法,能够高效合成l-PPT。