A computational strategy for altering an enzyme in its cofactor preference to NAD(H) and/or NADP(H)
A computational strategy for altering an enzyme in its cofactor preference to NAD(H) and/or NADP(H)
复制标题
改变酶对 NAD(H) 和/或 NADP(H) 的辅因子偏好的计算策略
DOI：
10.1111/febs.13282
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发表时间：
2015-06-01
期刊：
影响因子：
5.4
通讯作者：
Wei, Dongzhi
中科院分区：
文献类型：
--
作者：
Cui, Dongbing;Zhang, Lujia;Wei, Dongzhi
辅酶工程，特别是改变辅酶特异性，是近十多年来的研究热点。在本研究中，一种新的计算策略，增强酶和辅酶之间的氢键相互作用的开发和利用，以改变辅酶的偏好。这种新的计算策略只需要目标酶的结构。不需要其他同源酶来实现某种酶的辅酶偏好的改变。使用我们的新策略，Gox 2181从表现出完整的NADPH偏好重建为表现出对NADH和NADPH的双辅因子特异性。结构指导的Gox 2181突变体的设计在硅片和分子动力学模拟进行评估的强度的酶和辅酶NADPH之间的氢键相互作用。选择三个表现出高结构稳定性和对NADH/NADPH的结构相容性的Gox 2181突变体用于实验确认。Gox 2181-Q20 R和D43 S突变体中，以NADPH为辅酶的酶活最高，比野生型酶的酶活更高。此外，等温滴定量热分析进一步验证了Gox 2181 Q20 R &D43 S能够与NADPH相互作用，而野生型酶不能。这种新的计算策略代表了一种有见地的方法来改变目标enzymes.DatabaseModel数据库的辅助因子的偏好已经存放在蛋白质模型数据库的登录号为PM 0079165，PM 0079166，PM 0079167，PM 0079168和PM 0079169。
Coenzyme engineering, especially for altered coenzyme specificity, has been a research hotspot for more than a decade. In the present study, a novel computational strategy that enhances the hydrogen-bond interaction between an enzyme and a coenzyme was developed and utilized to alter the coenzyme preference. This novel computational strategy only required the structure of the target enzyme. No other homologous enzymes were needed to achieve alteration in the coenzyme preference of a certain enzyme. Using our novel strategy, Gox2181 was reconstructed from exhibiting complete NADPH preference to exhibiting dual cofactor specificity for NADH and NADPH. Structure-guided Gox2181 mutants were designed in silico and molecular dynamics simulations were performed to evaluate the strength of hydrogen-bond interactions between the enzyme and the coenzyme NADPH. Three Gox2181 mutants displaying high structure stability and structural compatibility to NADH/NADPH were chosen for experimental confirmation. Among the three Gox2181 mutants, Gox2181-Q20R&D43S showed the highest enzymatic activity by utilizing NADPH as its coenzyme, which was even better than the wild-type enzyme. In addition, isothermal titration calorimetry analysis further verified that Gox2181Q20R&D43S was able to interact with NADPH but the wild-type enzyme could not. This novel computational strategy represents an insightful approach for altering the cofactor preference of target enzymes.DatabaseModel data have been deposited in the Protein Model Database database under the accession numbers PM0079165, PM0079166, PM0079167, PM0079168 and PM0079169.