Key Laboratory of Molecular Biophysics, Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China.
Int J Mol Sci. 2023 May 11;24(10):8581. doi: 10.3390/ijms24108581.
Innovations in biocatalysts provide great prospects for intolerant environments or novel reactions. Due to the limited catalytic capacity and the long-term and labor-intensive characteristics of mining enzymes with the desired functions, de novo enzyme design was developed to obtain industrial application candidates in a rapid and convenient way. Here, based on the catalytic mechanisms and the known structures of proteins, we proposed a computational protein design strategy combining de novo enzyme design and laboratory-directed evolution. Starting with the constructed using a quantum-mechanical approach, the theoretical enzyme-skeleton combinations were assembled and optimized via the Rosetta "inside-out" protocol. A small number of designed sequences were experimentally screened using SDS-PAGE, mass spectrometry and a qualitative activity assay in which the designed enzyme 1a8uD exhibited a measurable hydrolysis activity of 24.25 ± 0.57 U/g towards p-nitrophenyl octanoate. To improve the activity of the designed enzyme, molecular dynamics simulations and the RosettaDesign application were utilized to further optimize the substrate binding mode and amino acid sequence, thus keeping the residues of intact. The redesigned lipase 1a8uD-M8 displayed enhanced hydrolysis activity towards p-nitrophenyl octanoate-3.34 times higher than that of 1a8uD. Meanwhile, the natural skeleton protein (PDB entry 1a8u) did not display any hydrolysis activity, confirming that the hydrolysis abilities of the designed 1a8uD and the redesigned 1a8uD-M8 were devised from scratch. More importantly, the designed 1a8uD-M8 was also able to hydrolyze the natural middle-chained substrate (glycerol trioctanoate), for which the activity was 27.67 ± 0.69 U/g. This study indicates that the strategy employed here has great potential to generate novel enzymes exhibiting the desired reactions.
生物催化剂的创新为耐受不良环境或新颖反应提供了广阔的前景。由于具有所需功能的酶的挖掘具有有限的催化能力和长期的、劳动密集型的特点，因此开发了从头酶设计方法，以便快速便捷地获得工业应用候选物。在这里，我们基于催化机制和已知的蛋白质结构，提出了一种将从头酶设计与实验室定向进化相结合的计算蛋白质设计策略。从使用量子力学方法构建的理论蛋白质骨架开始，通过 Rosetta“从内到外”的方案对理论酶-骨架组合进行组装和优化。通过 SDS-PAGE、质谱和定性活性测定实验筛选少量设计序列，其中设计的酶 1a8uD 对 p-硝基苯辛酸酯表现出可测量的水解活性，为 24.25±0.57 U/g。为了提高设计酶的活性，利用分子动力学模拟和 RosettaDesign 应用程序进一步优化了底物结合模式和氨基酸序列，从而保持了残基的完整性。重新设计的脂肪酶 1a8uD-M8 对 p-硝基苯辛酸酯-3 的水解活性提高了 3.34 倍，高于 1a8uD。同时，天然骨架蛋白（PDB 条目 1a8u）没有显示任何水解活性，这证实了设计的 1a8uD 和重新设计的 1a8uD-M8 的水解能力是从头设计的。更重要的是，设计的 1a8uD-M8 还能够水解天然中链底物（甘油三辛酸酯），其活性为 27.67±0.69 U/g。这项研究表明，这里采用的策略具有产生具有所需反应的新型酶的巨大潜力。
