丙二酰辅酶A是生物合成许多重要聚酮化合物的主要组成部分,以及脂肪酸衍生的化合物,比如生物燃料。大肠杆菌,gultamicum棒状杆菌,和酿酒酵母最近已被设计用于此类化合物的生物合成。然而,开发的工艺和菌株往往生产力不足。在目前的研究中,我们使用酶工程方法来改善乙酰辅酶A与ACC的结合。我们产生了不同的突变,计算了影响,它报告了三个突变,也就是说,S343A,T347W,和S350W,显著提高底物结合。分子对接研究揭示了与野生型相比改变的结合网络。在突变体中,额外的相互作用稳定乙酰辅酶A内尾的结合。使用分子模拟,稳定性,紧密度,氢键,和蛋白质运动被估计,揭示了仅突变体拥有的不同动态特性,而野生型则没有。通过使用无结合能(BFE)方法进一步验证了研究结果,这表明这些突变是有利的取代。据报道,野生型的总BFE为-52.66±0.11kcal/mol,S343A突变体为-55.87±0.16kcal/mol,-60.52±0.25kcal/mol对于T347W突变体,S350W突变体为-59.64±0.25kcal/mol。这表明底物的结合由于诱导的突变而增加,并且强烈证实对接结果。总之,这项研究提供了有关底物结合的必要热点残留物的信息,可用于工业过程中的应用。
Malonyl-CoA serves as the main building block for the biosynthesis of many important polyketides, as well as fatty acid-derived compounds, such as biofuel. Escherichia coli, Corynebacterium gultamicum, and Saccharomyces cerevisiae have recently been engineered for the biosynthesis of such compounds. However, the developed processes and strains often have insufficient productivity. In the current study, we used enzyme-engineering approach to improve the binding of acetyl-CoA with ACC. We generated different mutations, and the impact was calculated, which reported that three mutations, that is, S343A, T347W, and S350W, significantly improve the substrate binding. Molecular docking investigation revealed an altered binding network compared to the wild type. In mutants, additional interactions stabilize the binding of the inner tail of acetyl-CoA. Using molecular simulation, the stability, compactness, hydrogen bonding, and protein motions were estimated, revealing different dynamic properties owned by the mutants only but not by the wild type. The findings were further validated by using the binding-free energy (BFE) method, which revealed these mutations as favorable substitutions. The total BFE was reported to be -52.66 ± 0.11 kcal/mol for the wild type, -55.87 ± 0.16 kcal/mol for the S343A mutant, -60.52 ± 0.25 kcal/mol for T347W mutant, and -59.64 ± 0.25 kcal/mol for the S350W mutant. This shows that the binding of the substrate is increased due to the induced mutations and strongly corroborates with the docking results. In sum, this study provides information regarding the essential hotspot residues for the substrate binding and can be used for application in industrial processes.