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Structural and Functional Analysis of a Lytic Polysaccharide Monooxygenase Important for Efficient Utilization of Chitin in Cellvibrio japonicus

Lookup NU author(s): Dr Lucy Crouch

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Abstract

Cellvibrio japonicus is a Gram-negative soil bacterium that is primarily known for its ability to degrade plant cell wall polysaccharides through utilization of an extensive repertoire of carbohydrate-active enzymes. Several putative chitin-degrading enzymes are also found among these carbohydrate-active enzymes, such as chitinases, chitobiases, and lytic polysaccharide monooxygenases (LPMOs). In this study, we have characterized the chitin-active LPMO, CjLPMO10A, a tri-modular enzyme containing a catalytic family AA10 LPMO module, a family 5 chitin-binding module, and a C-terminal unclassified module of unknown function. Characterization of the latter module revealed tight and specific binding to chitin, thereby unraveling a new family of chitin-binding modules (classified as CBM73). X-ray crystallographic elucidation of the CjLPMO10A catalytic module revealed that the active site of the enzyme combines structural features previously only observed in either cellulose or chitin-active LPMO10s. Analysis of the copper-binding site by EPR showed a signal signature more similar to those observed for cellulose-cleaving LPMOs. The full-length LPMO shows no activity toward cellulose but is able to bind and cleave both alpha- and beta-chitin. Removal of the chitin-binding modules reduced LPMO activity toward alpha-chitin compared with the full-length enzyme. Interestingly, the full-length enzyme and the individual catalytic LPMO module boosted the activity of an endochitinase equally well, also yielding similar amounts of oxidized products. Finally, gene deletion studies show that CjLPMO10A is needed by C. japonicus to obtain efficient growth on both purified chitin and crab shell particles.


Publication metadata

Author(s): Forsberg Z, Nelson CE, Dalhus B, Mekasha S, Loose JSM, Crouch LI, Rohr AK, Gardner JG, Eijsink VGH, Vaaje-Kolstad G

Publication type: Article

Publication status: Published

Journal: Journal of Biological Chemistry

Year: 2016

Volume: 291

Issue: 14

Pages: 7300-7312

Print publication date: 01/04/2016

Online publication date: 08/02/2016

Acceptance date: 01/01/1900

ISSN (print): 0021-9258

ISSN (electronic): 1083-351X

Publisher: The American Society for Biochemistry and Molecular Biology, Inc.

URL: http://dx.doi.org/10.1074/jbc.M115.700161

DOI: 10.1074/jbc.M115.700161


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