Origin, evolution, and divergence of plant class C GH9 endoglucanases

Abstract

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.Electronic supplementary materialThe online version of this article (10.1186/s12862-018-1185-2) contains supplementary material, which is available to authorized users.

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

“…The microfibrillar structure of cellulose is constituted and strengthened by islands of hydrogen-bonded inter-glucan chains. These microcrystalline regions ( I α , I β ) render cellulose chemically inert and recalcitrant to most physical stressors, an attribute that is desirable to land plants (xylem, phloem), sporulating bacteria and fungi, and quorum sensing by microbial biofilms [ 1 – 8 ]. Most organisms (bacteria, fungi, protists) possess enzymes (oxidoreductases, EC 1.x.y.z; transferases, EC 2.x.y.z; hydrolases, EC 3.x.y.z) that can cleave cellulose into physiologically relevant oligo- and mono-saccharides (RXNs 1 – 3 ) [ 2 , 9 – 16 ].…”

“…Glycoside hydrolase 9 (GH9) endoglucanases ( EC 3.2.1.4) hydrolytically cleave the β (1 → 4)-glycoside linkage between contiguous ( D )-glucopyranose residues and accomplish this with the aid of one or more carbohydrate binding modules (CBMs). Detailed phylogenetics analysis and molecular dating has shown that GH9 (≅480 AA ) is very well conserved amongst taxa and has been so for ≈3000 Mya [ 8 , 17 ]. The presence of active site residues in GH9 further imply that catalysis of crystalline cellulose proceeds by a relatively unchanged generic acid-base mechanism and may deploy aspartic ( D ) and/or glutamic ( E ) acids as alternating proton donors/acceptors.…”

“…Carbohydrate-binding modules (CBMs) or carbohydrate-binding domains (CBDs) form distinct subsequences in eukaryotes (plants, CBM49; yeast, CBM54), protists ( Dictyostelium discoideum , CBM8), fungi (CBM1), and bacteria (CBMs 2-4) [ 8 , 17 , 18 ]. Most CBMs are separated by linkers (<100 AA ) from the GH domain(s) and vary in length (≈40 − 200 AA ), number, position (N-, C-termini, central), substrate affinity, and contribution to catalysis [ 8 , 17 – 41 ].…”

“…Carbohydrate-binding modules (CBMs) or carbohydrate-binding domains (CBDs) form distinct subsequences in eukaryotes (plants, CBM49; yeast, CBM54), protists ( Dictyostelium discoideum , CBM8), fungi (CBM1), and bacteria (CBMs 2-4) [ 8 , 17 , 18 ]. Most CBMs are separated by linkers (<100 AA ) from the GH domain(s) and vary in length (≈40 − 200 AA ), number, position (N-, C-termini, central), substrate affinity, and contribution to catalysis [ 8 , 17 – 41 ]. For example, GH9 endoglucanases from vascular land plants possess a unique subpopulation of CBM49-encompassing crystalline cellulose-digesting enzymes (class C) in addition to the amorphous cellulose cleaving subsets (classes A and B) [ 17 , 18 , 42 – 44 ].…”

“…For example, GH9 endoglucanases from vascular land plants possess a unique subpopulation of CBM49-encompassing crystalline cellulose-digesting enzymes (class C) in addition to the amorphous cellulose cleaving subsets (classes A and B) [ 17 , 18 , 42 – 44 ]. The presence of one or more CBMs may also extend the range of substrates of GH9 enzymes to include complex heteropolymeric moieties (chitin, CBM5, 12, 14, 18, 33; polygalactouronic acid, CBM32; lipopolysaccharide/lipoteichoic acid, CBM39) [ 8 , 17 , 19 , 35 – 41 ]. The precise mechanism(s) by which CBM-mediated catalysis proceeds is(are) debatable with several plausible explanations for the observed kinetic data [ 20 – 34 ].…”