生物学杂志

生物学杂志 ›› 2025, Vol. 42 ›› Issue (6): 1-.doi: 10.3969/j.issn.2095-1736.2025.06.001

• 研究报告 •    下一篇

自由能计算指导的cyst（e）inase的理性设计

唐梦佳1， 夏志勇1， 张　倩2， 樊　帅2， 张志斐1， 李小娜1， 杨兆勇2   

  1. 1. 华北理工大学 药学院， 唐山 063000；
    2. 中国医学科学院 北京协和医学院医药生物技术研究所， 北京 100050
  • 出版日期:2025-12-18 发布日期:2025-12-19
  • 通讯作者: 李小娜，博士，教授，研究方向为药剂学，E-mail：lixiaona71@126.com；张志斐，硕士，教授，研究方向为药物分析，E-mail：zhangzhifeifei7208@163.com；李小娜和张志斐为共同通信作者
  • 作者简介:唐梦佳，硕士研究生，研究方向为微生物与生化药学，E-mail：tmjia151752@outlook.com
  • 基金资助:
    国家自然科学基金面上项目 (82373767）； 中国医学科学院医学与健康科技创新工程项目（2021-I2M-1-055）

Rational design of cyst（e）inase guided by free energy calculations

TANG Mengjia1， XIA Zhiyong1, ZHANG Qian2, FAN Shuai2, ZHANG Zhifei1,LI Xiaona1, YANG Zhaoyong2   

  1. 1. College of Pharmacy, North China University of Science and Technology, Tangshan 063000, China; 2. Institute of
    Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College,
    Beijing 100050, China
  • Online:2025-12-18 Published:2025-12-19

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摘要： 胱硫醚γ-裂解酶（CGL）的突变体cyst（e）inase能够有效降解胱氨酸（CSSC）和半胱氨酸（L-Cys），并通过破坏肿瘤细胞的氧化还原稳态展现出抗肿瘤活性，但目前仍未达到临床应用的要求。为了提高cyst（e）inase在降解CSSC和L-Cys方面的活性及其热力学稳定性，研究通过分析蛋白序列的保守性，聚焦保守性较高的氨基酸位点，结合虚拟位点饱和突变计算自由能，筛选出潜在的有益突变体。研究结果表明，针对119个位点进行虚拟饱和突变后，通过自由能计算筛选出32个潜在的有益突变体。实验验证表明，其中，8个突变体在对CSSC和L-Cys的活性上有所提高。其中，最优突变体S206I，与cyst（e）inase相比，对CSSC和L-Cys活性分别提高了40%和60%，kcat/Km分别提高了23%和230%，Tm值提高了1.89 ℃。研究通过简便高效的设计策略，获得了具有活性和稳定性都提高的突变体，为后续酶制剂药物的改造提供了重要的示范。

关键词: 胱硫醚γ-裂解酶, 理性设计, 自由能计算, 序列保守性, 酶动力学

Abstract: Cystathionine γ-lyase (CGL) mutant cyst(e)inase effectively degrades cystine (CSSC) and cysteine (L-Cys), exhibiting antitumor activity through redox homeostasis disruption in tumor cells. However, its efficacy has not yet met the requirements for clinical application. To enhance the catalytic activity and thermodynamic stability of cyst(e)inase for CSSC and L-Cys degradation, this study focused on evolutionarily conserved amino acid residues using virtual site-saturation mutagenesis coupled with free energy calculations to identify potential beneficial mutants. The screening of 119 amino acid sites identified 32 promising mutants through free energy analysis. Experimental validation revealed that 8 mutants exhibited enhanced activity toward CSSC and L-Cys. Among these, the top-performing mutant, S206I, demonstrated 40% and 60% higher activity against CSSC and L-Cys, respectively, accompanied by 23% and 230% kcat/Km improvements. Additionally, its thermal stability (Tm) was elevated by 1.89 ℃. This study employed an efficient and straightforward design strategy, produced enhanced mutants with enhanced activity and stability, establishing a framework for subsequent therapeutic enzyme engineering.

Key words: cystathion γ-lyase, rational design, free energy calculation, sequence conservatism, enzyme kinetics

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