Abstract
Modular glycoside hydrolases that attack recalcitrant polymers generally contain noncatalytic carbohydrate-binding modules (CBMs), which play a critical role in the action of these enzymes by localizing the appended catalytic domains onto the surface of insoluble polysaccharide substrates. Type B CBMs, which recognize single polysaccharide chains, display ligand specificities that are consistent with the substrates hydrolyzed by the associated catalytic domains. In enzymes that contain multiple catalytic domains with distinct substrate specificities, it is unclear how these different activities influence the evolution of the ligand recognition profile of the appended CBM. To address this issue, we have characterized the properties of a family 11 CBM (CtCBM11) in Clostridium thermocellum Lic26A-Cel5E, an enzyme that contains GH5 and GH26 catalytic domains that display beta-1,4- and beta-1,3-1,4-mixed linked endoglucanase activity, respectively. Here we show that CtCBM11 binds to both beta-1,4- and beta-1,3-1,4-mixed linked glucans, displaying K(a) values of 1.9 x 10(5), 4.4 x 10(4), and 2 x 10(3) m(-1) for Glc-beta1,4-Glc-beta1,4-Glc-beta1,3-Glc, Glc-beta1,4-Glc-beta1,4-Glc-beta1,4-Glc, and Glc-beta1,3-Glc-beta1,4-Glc-beta1,3-Glc, respectively, demonstrating that CBMs can display a preference for mixed linked glucans. To determine whether these ligands are accommodated in the same or diverse sites in CtCBM11, the crystal structure of the protein was solved to a resolution of 1.98 A. The protein displays a beta-sandwich with a concave side that forms a potential binding cleft. Site-directed mutagenesis revealed that Tyr(22), Tyr(53), and Tyr(129), located in the putative binding cleft, play a central role in the recognition of all the ligands recognized by the protein. We propose, therefore, that CtCBM11 contains a single ligand-binding site that displays affinity for both beta-1,4- and beta-1,3-1,4-mixed linked glucans.
Highlights
The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ
This report reveals that CtCBM11 binds preferentially to -1,3–1,4 glucans while displaying considerable affinity for -1,4-linked glucose polymers and no affinity for -1,3 glucans
This ligand specificity reflects the substrate specificity of the associated GH26 and GH5 catalytic modules that act on -1,3–1,4- or -1,4-glucans, respectively
Summary
Protein Expression and Purification—To express CtCBM11 in Escherichia coli, the region of the Lic26A-Cel5A gene (lic26A-cel5A) encoding the internal family 11 CBM was amplified from C. thermocellum YS genomic DNA using the thermostable DNA polymerase pFU Turbo (Stratagene). Binding to Insoluble Polysaccharides—Qualitative assessment of CtCBM11 binding to Avicel and acid-swollen cellulose was carried out as follows: 30 g of protein in 50 mM Tris-HCl buffer, pH 7.5, containing 0.05% (v/v) Tween 20 and 5 mM CaCl2 (Buffer A) were mixed with 1 mg of ligand in a final reaction volume of 200 l. The molar concentration of CtCBM11-binding sites present in the polysaccharide ligands was determined as described previously [11]. Iterative model building with TURBO [27], together with refinement in REFMAC5 [28] and incorporation of the CtCBM11 primary sequence, resulted in a final model with R ϭ 19.3% (Rfree ϭ 23.2%) This final model includes 170 amino acid residues (five of which are selenomethionines), two calcium ions, two sulfate, and 182 water molecules.
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