腈水合酶
组氨酸
金属蛋白
化学
激活剂(遗传学)
活动站点
催化作用
酰胺
蛋白质工程
组合化学
酶
生物化学
受体
作者
Jidong Shen,Xue Cai,Ming Wang,Zhi‐Qiang Liu,Yu‐Guo Zheng
标识
DOI:10.1002/biot.202100103
摘要
Abstract Background Nitrile hydratase (NHase), was an excellent biocatalyst for the synthesis of amide compounds. NHase was typical heterodimeric metalloprotein, required of the assistance of activator for active expressions. In this work, we found a special Co‐NHase HBA from Caldalkalibacillus thermarum , which had the ability of post‐translational self‐modification and could incorporate Co 2+ into the catalytic center in the absence of activator. Method and Results We simulated the movement of Co 2+ in silico and established a hypothetical model to predict the Co 2+ incorporation efficiency (X Co ) of NHases. According to the simulation results, NHase mutants with different positive charge distribution were constructed. Compared with wild‐type, the Co 2+ incorporation efficiency of K1 (M10K) was increased by 2.1‐fold from 0.36 to 0.76, and the specific activity was increased by 3.2‐fold from 136.3 to 432.0 U mg ‐1 , while mutant K1H1 (M10K, D11H) and K2H2 (M10K, D11H, E20K, N21H) lost the ability of post‐translation self‐modification. Conclusions and Implications The interactions of positively charged residues near the catalytic center, such as lysine with strong electrostatic repulsive interaction, arginine with weak electrostatic repulsive interaction and histidine with metal affinity, could limit the free diffusion of Co 2+ in NHase and affect the efficiency of post‐translational self‐modification. This work also provided an effective strategy for protein engineering of NHases and other metalloenzymes.
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