Structural insights into the thermostability mechanism of a nitrile hydratase from Caldalkalibacillus thermarum by comparative molecular dynamics simulation

Nitrile hydratase (NHase), an excellent bio-catalyst for the synthesis of amide compounds, was composed of two heterologous subunits. A thermoalkaliphilic NHase NHCTA1 (Tm = 71.3 degrees C) obtained by in silico screening in our study exhibited high flexibility of alpha-subunit but excellent thermostability, as opposed to previous examples. To gain a deeper structural insight into the thermostability of NHCTA1, comparative molecular dynamics simulation of NHCTA1 and reported NHases was carried out. By comparison, we speculated that beta-subunit played a key role in adjusting the flexibility of alpha-subunit and the different conformations of linker in "alpha 5-helix-coil ring" supersecondary structure of beta-subunit can affect the interaction between beta-subunit and alpha-subunit. Mutant NHCTA1-alpha C-6 with a random coil linker and mutant NHCTA1-alpha beta gamma with a truncated linker were therefore constructed to understand the impact on NHCTA1 thermostability by varying the supersecondary structure. The varied thermostability of NHCTA1-alpha C-6 and NHCTA1-alpha beta gamma (Tm-alpha 6C = 74.4 degrees C, Tm-alpha beta gamma = 65.6 degrees C) verified that the flexibility of alpha-subunit adjusted by beta-subunit was relevant to the stability of NHCTA1. This study gained an insight into the NNHCTA1 thermostability by virtual dynamics comparison and experimental studies without crystallization, and this approach could be applied to other industrial-important enzymes.