The cellulase 12A from the hyperthermophile Thermotogo maritima (TmCel12A) is an ideal candidate to study the thermostability and the catalytic mechanism of cellulases, which are important for industries such as cellulosic ethanol production and food processing. The Optimal temperature of the wild-type TmCel12A is 85 °C and the turnover rate (kcat) is measured as 791 ± 16 s-1. Recent findings showed that a surface loop mutation Y61G was observed to not only increase the hydrolysis rate (with kcat up to 1155 ± 42 s-1), but also extend the half-life time of the enzyme at high temperature. Here, we explored the hydrolysis reaction and the thermostability mechanism of TmCel12A using free energy simulation with umbrella sampling and quantum mechanical/molecular mechanical (QM/MM) potentials, and molecular dynamics simulation with dynamic correlation network analysis, respectively. The free energy barriers for glycosylation and deglycosylation are calculated to be 22.5 ± 0.4 and 24.5 ± 0.7 kcal/mol at 85 °C. The barrier for deglycosylation is found to decrease with increasing temperature or as a result of the Y61G mutation. A hydrophobic cluster constructed by Y61, W176, V62, and L144 in the binding pocket is observed to play a pivotal role in modulating thermostability as well as the hydrolysis capability of TmCel12A. It stabilizes the apo enzyme at high temperature, however, impedes the substrate binding. Y61G mutation disturbs the hydrophobic cluster via replacing the π−π interaction between Y61 and W176 with the cation-π interaction between R60 and W176. The dynamic correlation network analysis indicates that Y61G mutation makes the enzyme more rigid and more extended via altering the amino acid communities at the hinge part of the enzyme. Early hypothesis that Y61G may accelerate the products releasing has been confirmed by our simulations too. These findings provide a new way to perform enzyme design to improve the catalysis rate and the thermostability of cellulases similar to TmCel12A.

The hyper-thermophilic and catalytic mechanism of extremophile cellulase TmCel12A

256th National Meeting (2018)

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