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Insights into the mechanism(s) of digestion of crystalline cellulose by plant class C GH9 endoglucanases.

ABSTRACT: Biofuels such as ?-valerolactone, bioethanol, and biodiesel are derived from potentially fermentable cellulose and vegetable oils. Plant class C GH9 endoglucanases are CBM49-encompassing hydrolases that cleave the ? (1???4) glycosidic linkage of contiguous D-glucopyranose residues of crystalline cellulose. Here, I analyse 3D-homology models of characterised and putative class C enzymes to glean insights into the contribution of the GH9, linker, and CBM49 to the mechanism(s) of crystalline cellulose digestion. Crystalline cellulose may be accommodated in a surface groove which is imperfectly bounded by the GH9_CBM49, GH9_linker, and linker_CBM49 surfaces and thence digested in a solvent accessible subsurface cavity. The physical dimensions and distortions thereof, of the groove, are mediated in part by the bulky side chains of aromatic amino acids that comprise it and may also result in a strained geometry of the bound cellulose polymer. These data along with an almost complete absence of measurable cavities, along with poorly conserved, hydrophobic, and heterogeneous amino acid composition, increased atomic motion of the CBM49_linker junction, and docking experiements with ligands of lower degrees of polymerization suggests a modulatory rather than direct role for CBM49 in catalysis. Crystalline cellulose is the de facto substrate for CBM-containing plant and non-plant GH9 enzymes, a finding supported by exceptional sequence- and structural-homology. However, despite the implied similarity in general acid-base catalysis of crystalline cellulose, this study also highlights qualitative differences in substrate binding and glycosidic bond cleavage amongst class C members. Results presented may aid the development of novel plant-based GH9 endoglucanases that could extract and utilise potential fermentable carbohydrates from biomass. Graphical Abstract Crystalline cellulose digestion by plant class C GH9 endoglucanases - an in silico assessment of function.

SUBMITTER: Kundu S

PROVIDER: S-EPMC7385011 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Insights into the mechanism(s) of digestion of crystalline cellulose by plant class C GH9 endoglucanases.

Kundu Siddhartha S

Journal of molecular modeling 20190723 8

Biofuels such as γ-valerolactone, bioethanol, and biodiesel are derived from potentially fermentable cellulose and vegetable oils. Plant class C GH9 endoglucanases are CBM49-encompassing hydrolases that cleave the β (1 → 4) glycosidic linkage of contiguous D-glucopyranose residues of crystalline cellulose. Here, I analyse 3D-homology models of characterised and putative class C enzymes to glean insights into the contribution of the GH9, linker, and CBM49 to the mechanism(s) of crystalline cellul ...[more]

PMID: 31338614

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Project description:BackgroundGlycoside hydrolases of the GH9 family encode cellulases that predominantly function as endoglucanases and have wide applications in the food, paper, pharmaceutical, and biofuel industries. The partitioning of plant GH9 endoglucanases, into classes A, B, and C, is based on the differential presence of transmembrane, signal peptide, and the carbohydrate binding module (CBM49). There is considerable debate on the distribution and the functions of these enzymes which may vary in different organisms. In light of these findings we examined the origin, emergence, and subsequent divergence of plant GH9 endoglucanases, with an emphasis on elucidating the role of CBM49 in the digestion of crystalline cellulose by class C members.ResultsSince, the digestion of crystalline cellulose mandates the presence of a well-defined set of aromatic and polar amino acids and/or an attributable domain that can mediate this conversion, we hypothesize a vertical mode of transfer of genes that could favour the emergence of class C like GH9 endoglucanase activity in land plants from potentially ancestral non plant taxa. We demonstrated the concomitant occurrence of a GH9 domain with CBM49 and other homologous carbohydrate binding modules, in putative endoglucanase sequences from several non-plant taxa. In the absence of comparable full length CBMs, we have characterized several low strength patterns that could approximate the CBM49, thereby, extending support for digestion of crystalline cellulose to other segments of the protein. We also provide data suggestive of the ancestral role of putative class C GH9 endoglucanases in land plants, which includes detailed phylogenetics and the presence and subsequent loss of CBM49, transmembrane, and signal peptide regions in certain populations of early land plants. These findings suggest that classes A and B of modern vascular land plants may have emerged by diverging directly from CBM49 encompassing putative class C enzymes.ConclusionOur detailed phylogenetic and bioinformatics analysis of putative GH9 endoglucanase sequences across major taxa suggests that plant class C enzymes, despite their recent discovery, could function as the last common ancestor of classes A and B. Additionally, research into their ability to digest or inter-convert crystalline and amorphous forms of cellulose could make them lucrative candidates for engineering biofuel feedstock.

Project description:The soil bacterium Cytophaga hutchinsonii actively digests crystalline cellulose by a poorly understood mechanism. Genome analyses identified nine genes predicted to encode endoglucanases with roles in this process. No predicted cellobiohydrolases, which are usually involved in the utilization of crystalline cellulose, were identified. Chromosomal deletions were performed in eight of the endoglucanase-encoding genes: cel5A, cel5B, cel5C, cel9A, cel9B, cel9C, cel9E, and cel9F Each mutant retained the ability to digest crystalline cellulose, although the deletion of cel9C caused a modest decrease in cellulose utilization. Strains with multiple deletions were constructed to identify the critical cellulases. Cells of a mutant lacking both cel5B and cel9C were completely deficient in growth on cellulose. Cell fractionation and biochemical analyses indicate that Cel5B and Cel9C are periplasmic nonprocessive endoglucanases. The requirement of periplasmic endoglucanases for cellulose utilization suggests that cellodextrins are transported across the outer membrane during this process. Bioinformatic analyses predict that Cel5A, Cel9A, Cel9B, Cel9D, and Cel9E are secreted across the outer membrane by the type IX secretion system, which has been linked to cellulose utilization. These secreted endoglucanases may perform the initial digestion within amorphous regions on the cellulose fibers, releasing oligomers that are transported into the periplasm for further digestion by Cel5B and Cel9C. The results suggest that both cell surface and periplasmic endoglucanases are required for the growth of C. hutchinsonii on cellulose and that novel cell surface proteins may solubilize and transport cellodextrins across the outer membrane.The bacterium Cytophaga hutchinsonii digests crystalline cellulose by an unknown mechanism. It lacks processive cellobiohydrolases that are often involved in cellulose digestion. Critical cellulolytic enzymes were identified by genetic analyses. Intracellular (periplasmic) nonprocessive endoglucanases performed an important role in cellulose utilization. The results suggest a model involving partial digestion at the cell surface, solubilization and uptake of cellodextrins across the outer membrane by an unknown mechanism, and further digestion within the periplasm. The ability to sequester cellodextrins and digest them intracellularly may limit losses of soluble cellobiose to other organisms. C. hutchinsonii uses an unusual approach to digest cellulose and is a potential source of novel proteins to increase the efficiency of conversion of cellulose into soluble sugars and biofuels.

Dataset Information

Insights into the mechanism(s) of digestion of crystalline cellulose by plant class C GH9 endoglucanases.

Publications

Insights into the mechanism(s) of digestion of crystalline cellulose by plant class C GH9 endoglucanases.

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