两种高度相似的二合成酶的表征和工程揭示了一个区域选择性的反开关
Dan Ye1, Yi-Zhen Shao1, Wen-Rui Li1
1State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050, People's Republic of China.
Angewandte Chemie (International ed. in English)
|February 8, 2024
概括
研究人员在控制产品多样性的聚二烯合成酶 (STPSs) 中确定了关键活性部位残留物. 突变这些残留物切换了酶功能,使得各种碳化合物骨架的新型STPS的合理设计成为可能.
科学领域:
- 生物化学 生物化学
- 酶学 是一种酶学.
- 有机化学 有机化学
背景情况:
- 六二烯合成酶 (STPSs) 通过碳基离子循环生成各种碳化合物.
- 在STPS中,反化步骤对于结构多样性至关重要,但它们的区域选择性基础尚未得到充分研究.
研究的目的:
- 调查STPSs中反质子化区域选择性的分子基础.
- 分析两个相似的STPSs,Artabotrys hexapetalus (-) -cyperene合成酶 (AhCS) 和ishwarane合成酶 (AhIS) 之间的功能差异.
主要方法:
- 对 AhCS 和 AhIS 的比较分析.
- 同位素标记实验,以确定生殖A (GA) 上的质子化位点.
- 低温电子显微镜 (cryo-EM) 用于确定AhCS的结构.
- 结构引导的位点导向突变发生.
主要成果:
- AhCS和AhIS催化了不同的反应,分别产生 (-) - 烯和伊什瓦兰.
- 同位素标记显示了GA的C3 (AhCS) 和C6 (AhIS) 的质子化.
- 冷-EM检测发现了关键的活性部位残留物,区分AhCS和AhIS.
- 三种残留物 (L311-S419-C458/M311-V419-A458) 的突变发生成功地相互转换了AhCS和AhIS功能,作为C3/C6反质子开关.
结论:
- 确定了一组特定的活性部位残留物,这些残留物可以作为STPS中生殖基A复原区域选择性的开关.
- 证明了合理设计的潜力,并指导STPSs的进化,以创建新的基骨架.
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