Structural Dynamics and Catalytic Properties of a Multimodular Xanthanase

The precise catalytic strategies used for thebreakdown of the complex bacterial polysaccharide xanthan, anincreasingly frequent component of processed human food-stuffs, have remained a mystery. Here, we present character-ization of anendo-xanthanase fromPaenibacillus nanensis.Weshow that it is a CAZy family 9 glycoside hydrolase (GH9)responsible for the hydrolysis of the xanthan backbone capableof generating tetrameric xanthan oligosaccharides frompolysaccharide lyase family 8 (PL8) xanthan lyase-treatedxanthan. Three-dimensional structure determination reveals acomplex multimodular enzyme in which a catalytic (α/α)6barrel isflanked by an N-terminal“immunoglobulin-like”(Ig-like) domain (frequently found in GH9 enzymes) and by fouradditional C-terminal allβ-sheet domains that have very few homologues in sequence databases and at least one of whichfunctions as a new xanthan-binding domain, now termed CBM84. The solution-phase conformation and dynamics of the enzymein the native calcium-bound state and in the absence of calcium were probed experimentally by hydrogen/deuterium exchangemass spectrometry. Measured conformational dynamics were used to guide the protein engineering of enzyme variants withincreased stability in the absence of calcium; a property of interest for the potential use of the enzyme in cleaning detergents. Theability of hydrogen/deuterium exchange mass spectrometry to pinpoint dynamic regions of a protein under stress (e.g., removalof calcium ions) makes this technology a strong tool for improving protein catalyst properties by informed engineering.