生物学杂志 ›› 2021, Vol. 38 ›› Issue (5): 48-.doi: 10.3969/j.issn.2095-1736.2021.05.048

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同源蛋白序列比对探讨AmPR-10核酸酶活性机理 #br# #br# #br# #br# #br#

  

  1. 山西中医药大学 基础医学院, 晋中 030600
  • 出版日期:2021-10-18 发布日期:2021-10-21
  • 通讯作者: 张育敏,博士,教授,研究方向为中医药在再生医学中的应用,E-mail: 9985855@qq.com
  • 作者简介:王 珏,硕士,讲师,研究方向为分子生物学,基因工程,蛋白定向进化,E-mail: wj.happysmile@163.com
  • 基金资助:
    山西省自然科学基金面上项目(201901D111339); 山西省高等学校科技创新项目(2020L0407); 山西省科技厅应用基础研究项目(201801D121230);山西省高等学校教学改革创新项目(J2020244)

Exploring the mechanism of AmPR-10 nuclease activity based on the homologous protein sequence alignment

  1. School of Basic Medicine, Shanxi University of Chinese Medicine, Jinzhong 030600, China
  • Online:2021-10-18 Published:2021-10-21

摘要: 蒙古黄芪病程相关蛋白AmPR-10(Astragalus membranaceus pathogenesis-related protein-10)对黄芪生长发育及抗病机制有重要意义。研究检测出以大肠杆菌为宿主外源表达的AmPR-10具有核酸酶活性。将AmPR-10与其同源的7个已证明具有核酸酶活性的蛋白进行氨基酸序列比对,发现AmPR-10基因有4个高保守区段,分别是:14~24位、44~55位、64~70位及83~88位。其中第一段保守区与C-末端的α螺旋在空间上形成较强的疏水区以稳定蛋白空腔的结构,利于AmPR-10在抵御侵害时功能的发挥;另由分子对接和氨基酸单点突变发现第4段保守区的83位Tyr是AmPR-10具有核酸酶活性的关键位点,其突变会影响目标蛋白与底物结合的稳定性,而非保守区域140位Gly可作为基因进化的参考位点,当突变为Asn、Val及Trp时,AmPR-10与底物的结合自由能下降。以上结果对深入理解及优化AmPR-10的核酸酶活性具有理论指导意义。

关键词: 蒙古黄芪病程相关蛋白, 同源蛋白, 分子对接; 核酸酶活性

Abstract: The AmPR-10(Astragalus membranaceus pathogenesis-related protein-10)plays an important role in the development and disease resistance of Astragalus membranaceus. The results showed that the AmPR-10 expressed in E. coli could display nuclease activity. Comparing the amino acid sequence of AmPR-10 with its seven homologous proteins which had been proved to have nuclease activity, we found that there were four highly conserved regions in the AmPR-10 gene (V14-V24, V44-K55, T64-K70 and Y83-G88 ). The first conserved region and the C-terminal α-helix formed a strong hydrophobic region in space to stabilize the structure of protein cavity, which was conducive to the function of AmPR-10 in resisting invasion. In addition, by molecular docking and amino acid site mutation, the Tyr at position 83 in the fourth conserved region was discovered to be the key site for AmPR-10 nuclease activity, and its change would affect the stability of target protein binding with substrate, while the 140 Gly in the non-conservative region could be used as a reference site for gene evolution. When Gly was mutated to Asn, Val and Trp, the binding free energy was decreased. The above results have theoretical guiding significance for further understanding and optimizing of the nuclease activity of AmPR-10.

Key words: AmPR-10, homologous protein, molecular docking, nuclease activity

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