5-03: Structure dynamics guided biocatalyst improvement

Enzyme dynamics has recently been shown to be crucial for its function. However, limited research has been carried out to study the structure dynamics for cell wall degrading enzymes using experimental approaches. We have successfully employed the state-of-the-art HDX mass spectrometry platform to probe the structure dynamics of several cellulase and xylanase enzymes. HDX mass spectrometry explores the dynamics of different protein regions based on the hydrogen-deteorium exchange rate. HDX mass spectrometry revealed significant intrinsic dynamics for the cellulase and xylanase enzymes. Different regions of the enzymes are differentially stabilized in the apo enzyme. The substrate and inhibitor binding also induces differential structure dynamics changes, whereas the competitive inhibitors induce similar changes as those of substrates. Among the different enzymes, the substrate binding stabilized xylanase the most, and we thereby present xylanase as a model system. The comparison of substrate-binding enzymes revealed that xylohexaose can significantly stabilize the enzyme. Several regions including those near the reaction centres were significantly stabilized during the xylohexaose binding. As compared to xylohexaose, xylan induced relatively less protection in the enzyme. The structure relevance of the enzyme dynamics was discussed with reference to the three dimensional structure of the enzyme. In particular, comparative sequence analysis revealed that thermostable xylanases have unique stabilization mechanisms for several key regions indicated by HDX experiments. According to the results, we have carried out the enzyme modification to improve the thermostability. Overall, HDX mass spectrometry revealed strong dynamics-function relevance and such relevance can be explored for the future enzyme improvement.