工程NADH氧化酶用于氧化非天然辅因子的再生
Xueying Wang1, Yeyu Liu1, Yinghan Hu1
1Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Division of Biotechnology, No. 457, Zhongshan Road, Dalian, China, 116023, Dalian, CHINA.
Chembiochem : a European journal of chemical biology
|May 22, 2025
概括
研究人员设计了一种新型酶,将尼古丁胺细胞因子二核酸 (NCDH) 转化为NCD+. 这一突破使得非自然共因子在合成生物学和化学生物学中的新应用成为可能.
科学领域:
- 生物化学 生物化学
- 合成生物学 合成生物学
- 代谢工程是代谢工程.
背景情况:
- 尼古丁胺胺氨基二核酸 (NAD+) 和NADH是关键的还原共因子,但由于代谢交叉,它们的操纵是复杂的.
- 非自然的辅助因子,如尼古丁胺胺细胞因子二核酸 (NCD+) 提供了替代品,但产生NCD+用于氧化反应仍然是一个挑战.
研究的目的:
- 设计一种NADH氧化酶 (NOX) 酶,能够有效地将NCDH氧化为NCD+.
- 建立一种使用非自然辅因子系统生成NCD+的方法.
主要方法:
- 为了有利于NCDH,Enterococcus faecalis NADH氧化酶 (EfNOX) 的蛋白质工程.
- 局部定向的突变发生和对EfNOX变种的查.
- 生物化学测试以确定催化效率和选择性.
- 计算对接分析以了解基质偏好.
- 埃舍里希亚大肠杆菌的代谢工程利用NCD+/NCDH系统.
主要成果:
- 与野生类型相比,工程NOX突变 (例如,NOX-KRGT) 对NCDH氧化显著提高了催化效率 (14倍) 和选择性 (107倍).
- 突变导致了更窄的辅因子结合腔和更为正电荷的环境,有利于NCDH.
- 将工程NOX突变体与NCD+依赖的酸盐脱酶结合起来,使大肠杆菌能够使用酸盐作为唯一源来生长.
结论:
- 开发了一种高效和选择性的酶,用于将NCDH转化为NCD+.
- 证明了工程系统在微生物新陈代谢中实施非自然辅因子的实用性.
- 这项工作扩大了化学和合成生物学中非自然辅因子应用的工具包.
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