CBM3d, a Novel Subfamily of Family 3 Carbohydrate-Binding Modules Identified in Cel48A Exoglucanase of Cellulosilyticum ruminicola

Abstract

Previously, we found that exoglucanase Cel48A from Cellulosilyticum ruminicola H1 bound intensively to Avicel; however, no known carbohydrate-binding module (CBM) was observed in the protein. Bioinformatics suggested that a C-terminal fragment of 127 amino acids, named the Cellulosilyticum-specific paralogous module (CPM), could function in binding. CPM-appended proteins are all putative (hemi)cellulases from Cellulosilyticum spp. In the present work, we demonstrated that Cel48A without the CPM retained only exoglucanase activity and lost the Avicel-binding ability, while the isolated CPM exhibited a high affinity for Avicel. In addition, the CPM bound to chitin, but not to soluble polysaccharides, making it a type A CBM, which binds only insoluble polysaccharides. Phylogenetic analysis clustered the CPM and its homologs as a separate branch that was distantly related to CBM subfamilies 3a (28% identity), 3b (24% identity), and 3c (21% identity). Sequence alignment revealed distinct secondary structures of the new CBM 3 group, in particular, a conserved Pro66-Trp67 insert preceding strand ␤4, a deletion preceding strand ␤6, and incomplete strands ␤8 and ␤9. An alanine scan for six aromatic and three nonaromatic amino acid residues (D66, P66, and R111) by site-directed mutagenesis determined that Phe62, Pro66, Trp67, Tyr68, Arg111, and Trp117 were the functional residues for binding. Among them, Phe62, Pro66, and Trp67 were the newly determined key sites in the CPM for binding. Three-dimensional homolog modeling revealed two types of substrate-binding sites, planar and groove, in the CPM. Thus, a new subfamily, CBM family 3d, is proposed.

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

“…Some CBM families are classified into subfamilies based on key residues in the ligand binding site (e.g. CBM3 4 ) or topological structure of the ligand binding region (e.g. CBM2 5 ).…”

Biochemical and Mutational Analyses of a Multidomain Cellulase/Mannanase from Caldicellulosiruptor bescii

“…GH9 family enzymes have an (␣/␣) 6 -barrel fold, and this family of enzymes is known to contain members that exhibit different enzymatic activities, including endoglucanase (45), cellobiohydrolase (15), 1,4-␤-D-glucan glucohydrolase (43), ␤-glucosidase (40), and exo-␤-glucosaminidase (23). The GH5 family of enzymes have an (␤/␣) 8 -barrel fold, and this family also encodes diverse enzymatic activities, including endoglucanase (16), mannanase (20), mannosidase (20), xylanase (14), chitosanase (50), glucosidase (11), licheninase (25), fucosidase (61), galactanase (44), endoglycoceramidase (9), and mannan transglycosylase (6). The binding cleft in GH9 catalytic modules can hold six sugar molecules, among which four subsites (Ϫ4 to Ϫ1) are at the nonreducing end and the two remaining subsites are at the reducing end (ϩ1 to ϩ2) (45).…”

“…The CBM3 modules are divided into four subfamilies based on the difference of the key planar linear strip aromatic or polar amino acid residues on the substrate binding surface (52). The CBM3a subfamily is only found associated with scaffoldins, CBM3b is associated with free enzymes, CBM3c is mainly linked to GH9 catalytic modules, and CBM3d is a newly identified module from Cellulosilyticum ruminicola (8).…”

Fine-structural variance of family 3 carbohydrate-binding modules as extracellular biomass-sensing components ofClostridium thermocellumanti-σIfactors

“…12b). A binding site that resembles the proposed alternative site of 0267CBM3b was recently suggested by Cai et al (2011) in the CBM3d from the Cel48A exoglucanase of Cellulosilyticum ruminicola. The Cel48A CBM3d has 24% homology to the CBM3b family and has a proposed cellulose-binding site located at the far end of the bottom face of the module.…”