Wednesday, May 2, 2012: 10:00 AM
Waterbury Ballroom, 2nd fl (Sheraton New Orleans)
Hydrogen Deuterium Exchange mass spectrometry (HDX-MS) stands out as an efficient and high-throughput way to analyze protein dynamics among various platforms. Comprehensive HDX-MS analysis of several cell wall degrading enzymes revealed both new structural determinants for enzyme improvement and novel insights into enzyme structure-function relationship. In particular, we used Trichoderma reesei endo-β-1,4-xylanase (XYN1) as model hemicellulolytic enzyme for HDX-MS analysis, where several regions were identified as stabilized or destabilized upon ligand binding. Further site-directed mutagenesis enabled XYN1 with increased activity, heterologous expression compatibility, and stability. Besides serving as novel enzyme improvement platform, comparative HDX-MS analysis of XYN1 and Aspergillus niger EG1A has uncovered new relationship between catalysis and dynamics. Remarkable differential dynamics were identified for both enzymes although they are structurally highly similar. Compared to EG1A, XYN1 displayed highly intrinsic structure dynamics at both regional and global levels, as revealed by two complementary HDX-MS methods. The current findings have also been confirmed by the molecular simulations of enzyme dynamics at ps and ns levels. The results highlighted that intrinsic dynamics may not always underlie catalysis, and dynamics may have broader roles in substrate specificity and enzyme activity. Domain swapping between both enzymes has been carried out to verify the hypothesis and dissect the new aspects for dynamics-function relationship. Overall, our study highlighted that HDX-MS can serve as a powerful platform for rational design of biocatalysts and mechanistic studies. Novel enzymes with higher activity and better heterologous expression compatibility can be produced using this platform for many biofuel applications including CBP.