Department of Biotechnology, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Poznań, Poland.
Department of Crystal Chemistry and Crystal Physics, Faculty of Chemistry, Jagiellonian University, Krakow, Poland.
Protein Sci. 2023 Jun;32(6):e4647. doi: 10.1002/pro.4647.
The Escherichia coli enzyme EcAIII catalyzes the hydrolysis of L-Asn to L-Asp and ammonia. Using a nature-inspired mutagenesis approach, we designed and produced five new EcAIII variants (M200I, M200L, M200K, M200T, M200W). The modified proteins were characterized by spectroscopic and crystallographic methods. All new variants were enzymatically active, confirming that the applied mutagenesis procedure has been successful. The determined crystal structures revealed new conformational states of the EcAIII molecule carrying the M200W mutation and allowed a high-resolution observation of an acyl-enzyme intermediate with the M200L mutant. In addition, we performed structure prediction, substrate docking, and molecular dynamics simulations for 25 selected bacterial orthologs of EcAIII, to gain insights into how mutations at the M200 residue affect the active site and substrate binding mode. This comprehensive strategy, including both experimental and computational methods, can be used to guide further enzyme engineering and can be applied to the study of other proteins of medicinal or biotechnological importance.
大肠杆菌酶 EcAIII 催化 L-天冬酰胺水解生成 L-天冬氨酸和氨。本研究采用受自然启发的诱变方法，设计并生产了五种新型 EcAIII 变体（M200I、M200L、M200K、M200T 和 M200W）。通过光谱和晶体学方法对修饰后的蛋白质进行了表征。所有新型变体均具有酶活性，这证实了所应用的诱变程序是成功的。确定的晶体结构揭示了携带 M200W 突变的 EcAIII 分子的新构象状态，并允许对 M200L 突变体的酰基-酶中间产物进行高分辨率观察。此外，我们针对 25 种选定的 EcAIII 细菌同源物进行了结构预测、底物对接和分子动力学模拟，以深入了解 M200 残基的突变如何影响活性位点和底物结合模式。这种综合策略包括实验和计算方法，可以用于指导进一步的酶工程，并可应用于研究其他具有医学或生物技术重要性的蛋白质。
