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.gribble | carbohydrate degrading enzymes

Biochemical and Structural Characterizations of Two Dictyostelium Cellobiohydrolases from the Amoebozoa Kingdom Reveal a High Level of Conservation between Distant Phylogenetic Trees of Life

“…Continued structural and biochemical studies of these enzymes are essential to developing detailed structure-function relationships, given their importance. The GH7 CBHs from D. discoideum and D. purpureum are now the second and third known nonfungal GH7 enzyme structures, with the first one being from a salt water-inhabiting, wood-boring isopod, L. quadripunctata (22).…”

“…The structures of GH7 catalytic domains are known from 10 CBHs, Trichoderma reesei Cel7A (TreCel7A) (12)(13)(14), Heterobasidion irregulare Cel7A (HirCel7A) (15), Phanerochaete chrysosporium Cel7D (PchCel7D) (16), Talaromyces emersonii Cel7A (RemCel7A) (17), Trichoderma harzianum Cel7A (ThaCel7A) (18), Melanocarpus albomyces Cel7B (MalCel7B) (19), Aspergillus fumigatus Cel7A (AfuCel7A) (20), Humicola grisea var. thermoidea Cel7A (HgtCel7A) (21) Limnoria quadripunctata Cel7B (LquCel7B) (22), and Geotrichum candidum Cel7A (23), and three endoglucanases (EGs), T. reesei Cel7B (24), Humicola insolens Cel7B (25), and Fusarium oxysporum Cel7B (26). All GH7s share a ␤-jelly roll fold, with two antiparallel ␤-sheets packing face to face to form a curved ␤-sandwich.…”

The genome of the crustacean Parhyale hawaiensis, a model for animal development, regeneration, immunity and lignocellulose digestion

“…Apart from attracting research interest as an economically important food crop, this group of animals has been used to study developmental biology and the evolution of morphological diversity (for example with respect to Hox genes) (Martin et al, 2015; Averof and Patel, 1997; Liubicich et al, 2009; Pavlopoulos et al, 2009), stem cell biology (Konstantinides and Averof, 2014; Benton et al, 2014), innate immunity processes (Vazquez et al, 2009; Hauton, 2012) and recently the cellular mechanisms of regeneration (Konstantinides and Averof, 2014; Benton et al, 2014; Alwes et al, 2016). In addition, members of the Malacostraca, specifically both Amphipods and Isopods, are thought to be capable of 'wood eating' or lignocellulose digestion and to have microbiota-free digestive systems (King et al, 2010; Kern et al, 2013; Boyle and Mitchell, 1978; Zimmer et al, 2002). …”

Towards a molecular-level theory of carbohydrate processivity in glycoside hydrolases

“…This catalytically-active complex may resemble the theoretical model of the Michaelis complex of T. reesei Cel7A (PDB code 8CEL, Figure 2, bottom right). Immediately following the first catalytic step (Glycosylation), the cellobiose product resides in what we refer to as the 'Unprimed glycosyl-enzyme intermediate' (GEI) mode (seen in PDB structures 6CEL, 7CEL, 1Q2E, 1Z3W, 2RFY, 4HAP, 4IPM, and exemplified by the 7CEL structure in Figure 2, lower left frame) [21,44,63,69,70]. As with the Michaelis complex, no GEI crystal structure has been published to date for a GH Family 7 member.…”