How nature can exploit nonspecific catalytic and carbohydrate binding modules to create enzymatic specificity

Abstract

Noncatalytic carbohydrate binding modules (CBMs) are components of glycoside hydrolases that attack generally inaccessible substrates. CBMs mediate a two-to fivefold elevation in the activity of endoacting enzymes, likely through increasing the concentration of the appended enzymes in the vicinity of the substrate. The function of CBMs appended to exo-acting glycoside hydrolases is unclear because their typical endo-binding mode would not fulfill a targeting role. Here we show that the Bacillus subtilis exo-acting β-fructosidase SacC, which specifically hydrolyses levan, contains the founding member of CBM family 66 (CBM66). The SacC-derived CBM66 (BsCBM66) targets the terminal fructosides of the major fructans found in nature. The crystal structure of BsCBM66 in complex with ligands reveals extensive interactions with the terminal fructose moiety (Fru-3) of levantriose but only limited hydrophobic contacts with Fru-2, explaining why the CBM displays broad specificity. Removal of BsCBM66 from SacC results in a ∼100-fold reduction in activity against levan. The truncated enzyme functions as a nonspecific β-fructosidase displaying similar activity against β-2,1-and β-2,6-linked fructans and their respective fructooligosaccharides. Conversely, appending BsCBM66 to BT3082, a nonspecific β-fructosidase from Bacteroides thetaiotaomicron, confers exolevanase activity on the enzyme. We propose that BsCBM66 confers specificity for levan, a branched fructan, through an "avidity" mechanism in which the CBM and the catalytic module target the termini of different branches of the same polysaccharide molecule. This report identifies a unique mechanism by which CBMs modulate enzyme function, and shows how specificity can be tailored by integrating nonspecific catalytic and binding modules into a single enzyme.isothermal titration calorimetry | X-ray crystallography | prebiotics | biofuels | lectins C omplex carbohydrates represent a major nutrient for numerous microbial ecosystems, exemplified by bacterial and fungal communities established in the rumen and large bowel of mammals, where they play an important role in animal nutrition and human health, respectively (1, 2). It is also evident that these composite structures are of increasing industrial significance, particularly in the bioenergy and bioprocessing sectors (3). The enzymes that catalyze the degradation of these complex carbohydrates, primarily glycoside hydrolases but also polysaccharide lyases, are grouped into sequence-based families on the continuously updated CAZy database (4). Members of the same family display a common fold, and the catalytic mechanism and catalytic apparatus are also conserved (5). Substrate specificity within glycoside hydrolase families (GHs), however, can vary significantly (6). Glycanases that attack inaccessible substrates often contain noncatalytic carbohydrate binding modules (CBMs; see ref. 7 for review) that are also grouped into sequence-based families in the CAZy database. Against recalcitrant substrates, CBMs enhance the activit...

The Quaternary Structure of a Glycoside Hydrolase Dictates Specificity toward β-Glucans

“…The present characterization of SdGluc5_26A allows us to ascertain that its specificity finds its origin in the quaternary structure. Until this work, only a carbohydrate-binding module has previously been shown to be able to affect the substrate specificity of the appended catalytic domain (40). To further confirm the influence of the quaternary structural assembly on the substrate specificity of SdGluc5_26A, we produced the deletion mutant SdGluc5_26A⌬S38, devoid of the helix-turn motif interacting with residues of the substrate-binding cleft at the level of subsite Ϫ3.…”

Predicting Enzyme Adsorption to Lignin Films by Calculating Enzyme Surface Hydrophobicity

“…CBMs, divided into numerous families based on protein fold, display a variety of substrate specificities (47,48). In some cases a multivalent effect has been observed, where single cellulase enzymes contain multiple CBMs to increase association with target substrates (49,50).…”

Fungal lytic polysaccharide monooxygenases bind starch and β‐cyclodextrin similarly to amylolytic hydrolases

“…polysaccharides [18,19]. Similarly, starch-specific CBMs, which are assigned into CBM20 in the CAZy database [20,21], are reported to potentate the activity of fungal amylolytic enzymes, for example, a-amylases and glucoamylases on granular starch [22,23].…”