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Structural dynamics and catalytic properties of a multimodular xanthanase

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JournalACS Catalysis
DateAccepted/In press - 11 May 2018
DateE-pub ahead of print - 11 May 2018
DatePublished (current) - 6 Jul 2018
Issue number7
Volume8
Number of pages14
Pages (from-to)6021–6034
Early online date11/05/18
Original languageEnglish

Abstract

The precise catalytic strategies used for the breakdown of the complex bacterial polysaccharide xanthan, an increasingly frequent component of processed human foodstuffs, have remained a mystery. Here, we present characterization of an endo-xanthanase from Paenibacillus nanensis. We show that it is a CAZy family 9 glycoside hydrolase (GH9) responsible for the hydrolysis of the xanthan backbone capable of generating tetrameric xanthan oligosaccharides from polysaccharide lyase family 8 (PL8) xanthan lyase-treated xanthan. Three-dimensional structure determination reveals a complex multimodular enzyme in which a catalytic (α/α) 6 barrel is flanked by an N-terminal "immunoglobulin-like" (Ig-like) domain (frequently found in GH9 enzymes) and by four additional C-terminal all β-sheet domains that have very few homologues in sequence databases and at least one of which functions as a new xanthan-binding domain, now termed CBM84. The solution-phase conformation and dynamics of the enzyme in the native calcium-bound state and in the absence of calcium were probed experimentally by hydrogen/deuterium exchange mass spectrometry. Measured conformational dynamics were used to guide the protein engineering of enzyme variants with increased stability in the absence of calcium; a property of interest for the potential use of the enzyme in cleaning detergents. The ability of hydrogen/deuterium exchange mass spectrometry to pinpoint dynamic regions of a protein under stress (e.g., removal of calcium ions) makes this technology a strong tool for improving protein catalyst properties by informed engineering.

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© 2018 American Chemical Society. This is an author-produced version of the published paper. Uploaded in accordance with the publisher’s self-archiving policy. Further copying may not be permitted; contact the publisher for details

    Research areas

  • carbohydrate, enzyme, enzyme dynamics, enzyme stability, hydrogen/deuterium exchange mass spectrometry, xanthan

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