Abstract
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 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 Crystalline cellulose digestion by plant class C GH9 endoglucanases - an in silico assessment of function.
Highlights
The microfibrillar structure of cellulose is constituted and strengthened by islands of hydrogen-bonded inter-glucan chains
A pipeline comprising each step and the relevant data generated are presented as under the following steps: Step 0: Parameters were defined for protocols to minimise, equilibriate, and preliminarily characterise 3D models of plant class C Glycoside hydrolase 9 (GH9) endoglucanases and ligands of cellulose (Fig. 1, Tables 1 and 2) Step 1: The 3D fold of sequences of characterised and putative plant class C GH9 endoglucanases was determined (Figs. 1 and 2, Table 3; Supplementary Text 1)
Step 5: Structural homologues of selected characterised and putative class C enzymes were identified with a principal component analysis (PCA)-based clustering schema and analysed to derive insignts into the mechanism(s) of digesting crystalline cellulose by plant class C GH9 endoglucanases (Figs. 8 and 9, Table 9; Supplementary Text 10)
Summary
The microfibrillar structure of cellulose is constituted and strengthened by islands of hydrogen-bonded inter-glucan chains. These microcrystalline regions (Iα, Iβ) render cellulose. FC2; C2þg þ fe−; 2e−g⇌fC2 1⁄4 O; C2þ 1⁄4 Og ðRXN1Þ. Cn þ H iPO4⇋C−OPO3 þ Cn−1 ðRXN2Þ. Cn þ kH2O⇋ þ ðm1ÞðCn−iÞ þ kH2O⇋ðm2ÞðC2−5Þ þ kH2O⇋ðmnÞðC1Þ ðRXN3Þ 240 Page 2 of 24 Cn ≔ Glucan. C ≔ DðαÞ−glucopyranose phosphate i ∈ f1; 2; 3g. C2 ≔ Cellulose with degree of polymerization ðDP 1⁄4 2Þ
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