Identification of a novel family of carbohydrate-binding modules with broad ligand specificity

Abstract

Most enzymes that act on carbohydrates include non-catalytic carbohydrate-binding modules (CBMs) that recognize and target carbohydrates. CBMs bring their appended catalytic modules into close proximity with the target substrate and increase the hydrolytic rate of enzymes acting on insoluble substrates. We previously identified a novel CBM (CBMC5614-1) at the C-terminus of endoglucanase C5614-1 from an uncultured microorganism present in buffalo rumen. In the present study, that the functional region of CBMC5614-1 involved in ligand binding was localized to 134 amino acids. Two representative homologs of CBMC5614-1, sharing the same ligand binding profile, targeted a range of β-linked polysaccharides that adopt very different conformations. Targeted substrates included soluble and insoluble cellulose, β-1,3/1,4-mixed linked glucans, xylan, and mannan. Mutagenesis revealed that three conserved aromatic residues (Trp-380, Tyr-411, and Trp-423) play an important role in ligand recognition and targeting. These results suggest that CBMC5614-1 and its homologs form a novel CBM family (CBM72) with a broad ligand-binding specificity. CBM72 members can provide new insight into CBM-ligand interactions and may have potential in protein engineering and biocatalysis.

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

“…Plant class C enzymes share considerable structural homology with gram-positive and -negative bacterial GH9 members (Tables 1 , 2 , and 3 ; Supplementary Texts 1 and 2 ). Although these results for GH9 are not entirely unexpected, data from this study also supports the involvement of the linker and CBM49 in the catalysis of crystalline cellulose by plant class C enzymes (Table 3 ; Supplementary Texts 1 and 2 ) [ 8 , 17 , 20 – 34 , 59 , 60 ]. The inclusion of the N- and C-terminal linker, albeit at higher volumes ( V ∈ (8.0,100.0]) and the complete exclusion of CBM49 even amongst this small subset of class C enzymes suggest poor conservation of these segments (Figs.…”

“…2 ). The results (rmsd (template, x ) < 2 Ang ) suggest that the catalytic machinery for digesting crystalline may be conserved in plants and other non-plant taxa most notably bacteria (Table 3 ; Supplementary Text 1 ) [ 8 , 17 , 20 – 34 , 59 , 60 ]. The models also indicate that in addition to GH9, CBM49 and the linker (coverage = 89 − 94%) may partake in digesting crystalline cellulose (Table 3 ) [ 8 , 17 , 59 , 60 ].…”

“…The ability to cleave crystalline cellulose by plant class C members is dependent on the presence of CBM49 and may have evolved directly from non-plant taxa (≈500 Mya) [ 8 , 17 , 20 – 34 , 59 , 60 ]. An additional premise explored previously was that plant class C enzymes may not just predate but, could potentially diverge into classes A and B after CBM49 was excised during processing of the mature mRNA transcript [ 8 , 18 , 46 , 83 – 85 ].…”

“…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 ]. Most CBMs possess non-contiguous aromatic amino acids (tryptophan/phenylalanine/tyrosine) interspersed with amino acids with shorter side chains.…”

Genome sequencing and analysis of Talaromyces pinophilus provide insights into biotechnological applications

“…The most prevalent GH CAZyme classes were GH7, GH3, GH5, GH10-13 and GH31, which represent cellulases, endoxylanases and amylases, all of which are required for biomass degradation. The most abundant CBM family was CBM1, accounting for 43.8% of total CBMs, which are known to bind to crystalline cellulose and aid in its enzymatic hydrolysis 32 .…”