Enzyme structure dynamics of xylanase I from Trichoderma longibrachiatum

BACKGROUND: Enzyme dynamics has recently been shown to be crucial for structure-function relationship. Among various structure dynamics analysis platforms, HDX (hydrogen deuterium exchange) mass spectrometry stands out as an efficient and high-throughput way to analyze protein dynamics upon ligand binding. Despite the potential, limited research has employed the HDX mass spec platform to probe regional structure dynamics of enzymes. In particular, the technique has never been used for analyzing cell wall degrading enzymes. We hereby used xylanase as a model to explore the potential of HDX mass spectrometry for studying cell wall degrading enzymes. RESULTS: HDX mass spectrometry revealed significant intrinsic dynamics for the xylanase enzyme. Different regions of the enzymes are differentially stabilized in the apo enzyme. 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, which may be due to the insolubility of the substrate. The structure relevance of the enzyme dynamics was discussed with reference to the three dimensional structure of the enzyme. HDX mass spectrometry revealed strong dynamics-function relevance and such relevance can be explored for the future enzyme improvement. CONCLUSION: Ligand-binding can lead to the significant stabilization at both regional and global level for enzymes like xylanase. HDX mass spectrometry is a powerful high-throughput platform to identify the key regions protected during the ligand binding and to explore the molecular mechanisms of the enzyme function. The HDX mass spectrometry analysis of cell wall degrading enzymes has provided a novel platform to guide the rational design of enzymes

Structure Dynamics Guided Enzyme Improvement of ENDO-BETA-1, 4-XYLANASE I

Enzyme structure dynamics has recently been revealed to be essential for structure-function relationship. Among various structure dynamics analysis platforms, hydrogen deuterium exchange mass spectrometry stands as an efficient and high-throughput way to analyze protein dynamics upon ligand binding, protein folding, and enzyme catalysis. HDX-MS can be used to study the regional dynamics of proteins based on the m/z value or percentage of deuterium incorporation for the digested peptides in the HDX experiments. Various software packages have been developed to analyze HDX-MS data. However, for the accurate, enhanced, and explicit statistical analysis of HDX-MS data statistical analysis of software was developed as HDXanalyzer. The capability of HDX-MS analysis for the identification of enzyme structure dynamics was tested by using model catalysis endoxylanase A (XYN I) from Trichoderma longibrachiatum. The HDX data of XYN I revealed a highly dynamic personality of XYN I through the interaction with two substrates. The dynamic data which certainly restricts the targeted regions for the protein engineering efforts provided useful knowledge about the essential structural modifications for the catalysis of XYN I. The obtained knowledge was then employed for the engineering studies in order to improve the certain characteristics of XYN I protein. The high level stabilization of XYN I protein was gathered and the two highly active and moderately thermostable XYN I recombinants were developed based on the HDX-MS data which further confirmed the efficiency of the current strategy for the rational designs of catalytic proteins. A differential dynamics analysis of the two structurally similar catalysts was also performed through HDX-MS. The functionally and sequentially different but structurally highly similar XYN I and endoglucanase (Eg1A) enzymes revealed distinct structure dynamic characteristics. Compared to XYN I, Eg1A from Aspergillus niger indicated quite restricted structural motions. The data clearly postulated that the intrinsic dynamic modifications of during the enzymatic catalysis may not be the only driving force in all cases. In summary, the integration of the structure dynamics knowledge to the current biochemical and biophysical data of catalysts may provide novel insights to further enzyme improvement applications