title: Structural characterization of a unique marine animal family 7 cellobiohydrolase suggests a mechanism of cellulase salt tolerance.
abstract: Nature uses a diversity of glycoside hydrolase (GH) enzymes to convert polysaccharides to sugars. As lignocellulosic biomass deconstruction for biofuel production remains costly, natural GH diversity offers a starting point for developing industrial enzymes, and fungal GH family 7 (GH7) cellobiohydrolases, in particular, provide significant hydrolytic potential in industrial mixtures. Recently, GH7 enzymes have been found in other kingdoms of life besides fungi, including in animals and protists. Here, we describe the in vivo spatial expression distribution, properties, and structure of a unique endogenous GH7 cellulase from an animal, the marine wood borer Limnoria quadripunctata (LqCel7B). RT-quantitative PCR and Western blot studies show that LqCel7B is expressed in the hepatopancreas and secreted into the gut for wood degradation. We produced recombinant LqCel7B, with which we demonstrate that LqCel7B is a cellobiohydrolase and obtained four high-resolution crystal structures. Based on a crystallographic and computational comparison of LqCel7B to the well-characterized Hypocrea jecorina GH7 cellobiohydrolase, LqCel7B exhibits an extended substrate-binding motif at the tunnel entrance, which may aid in substrate acquisition and processivity. Interestingly, LqCel7B exhibits striking surface charges relative to fungal GH7 enzymes, which likely results from evolution in marine environments. We demonstrate that LqCel7B stability and activity remain unchanged, or increase at high salt concentration, and that the L. quadripunctata GH mixture generally contains cellulolytic enzymes with highly acidic surface charge compared with enzymes derived from terrestrial microbes. Overall, this study suggests that marine cellulases offer significant potential for utilization in high-solids industrial biomass conversion processes.
authors.affiliation: Centre for Novel Agricultural Products, Department of Biology, University of York, York YO10 5DD, United Kingdom.
authors.forename: Marcelo
authors.initials: M
authors.lastname: Kern
authors.forename: John E
authors.initials: JE
authors.lastname: McGeehan
authors.forename: Simon D
authors.initials: SD
authors.lastname: Streeter
authors.forename: Richard N A
authors.initials: RN
authors.lastname: Martin
authors.forename: Katrin
authors.initials: K
authors.lastname: Besser
authors.forename: Luisa
authors.initials: L
authors.lastname: Elias
authors.forename: Will
authors.initials: W
authors.lastname: Eborall
authors.forename: Graham P
authors.initials: GP
authors.lastname: Malyon
authors.forename: Christina M
authors.initials: CM
authors.lastname: Payne
authors.forename: Michael E
authors.initials: ME
authors.lastname: Himmel
authors.forename: Kirk
authors.initials: K
authors.lastname: Schnorr
authors.forename: Gregg T
authors.initials: GT
authors.lastname: Beckham
authors.forename: Simon M
authors.initials: SM
authors.lastname: Cragg
authors.forename: Neil C
authors.initials: NC
authors.lastname: Bruce
authors.forename: Simon J
authors.initials: SJ
authors.lastname: McQueen-Mason
mesh_terms: D000818:Animals; D056804:Biofuels; D018533:Biomass; D002480:Cellulase / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003445:Crustacea / Q000201:enzymology* / Q000235:genetics; D018360:Crystallography, X-Ray; D004064:Digestive System / Q000201:enzymology; D004789:Enzyme Activation / Q000502:physiology; D035901:Hypocrea / Q000201:enzymology; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D055049:Salt Tolerance / Q000502:physiology*; D012623:Seawater; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D056804:Biofuels; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase
keywords: carbohydrate degrading enzymes; gribble