Structural dynamics and catalytic properties of a multimodular xanthanase

TY - JOUR

T1 - Structural dynamics and catalytic properties of a multimodular xanthanase

AU - Moroz, Olga V.

AU - Jensen, Pernille F.

AU - McDonald, Sean P.

AU - McGregor, Nicholas

AU - Blagova, Elena

AU - Comamala, Gerard

AU - Segura, Dorotea R.

AU - Anderson, Lars

AU - Vasu, Santhosh M.

AU - Rao, Vasudeva P.

AU - Giger, Lars

AU - Sørensen, Trine Holst

AU - Monrad, Rune Nygaard

AU - Svendsen, Allan

AU - Nielsen, Jens E.

AU - Henrissat, Bernard

AU - Davies, Gideon J.

AU - Brumer , Harry

AU - Rand, Kasper

AU - Wilson, Keith S.

N1 - © 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

PY - 2018/7/6

Y1 - 2018/7/6

N2 - 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.

AB - 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.

KW - carbohydrate

KW - enzyme

KW - enzyme dynamics

KW - enzyme stability

KW - hydrogen/deuterium exchange mass spectrometry

KW - xanthan

UR - http://www.scopus.com/inward/record.url?scp=85047082081&partnerID=8YFLogxK

U2 - 10.1021/acscatal.8b00666

DO - 10.1021/acscatal.8b00666

M3 - Article

SN - 2155-5435

VL - 8

SP - 6021

EP - 6034

JO - ACS Catalysis

JF - ACS Catalysis

IS - 7

ER -