A Novel, Noncatalytic Carbohydrate-binding Module Displays Specificity for Galactose-containing Polysaccharides through Calcium-mediated Oligomerization

Cedric Y. Montanier,Pedro M. Coutinho,José A.M. Prates,Bernard Henrissat,Márcia A.S. Correia,Harry J. Gilbert,Arnaud Baslé,James E. Flint,Richard J. Lewis,Samuel J. Polizzi,Carlos M.G.A. Fontes,Yanping Zhu,Lauren S. McKee

doi:10.1074/jbc.m110.217372

Abstract

The enzymic degradation of plant cell walls plays a central role in the carbon cycle and is of increasing environmental and industrial significance. The catalytic modules of enzymes that catalyze this process are generally appended to noncatalytic carbohydrate-binding modules (CBMs). CBMs potentiate the rate of catalysis by bringing their cognate enzymes into intimate contact with the target substrate. A powerful plant cell wall-degrading system is the Clostridium thermocellum multienzyme complex, termed the "cellulosome." Here, we identify a novel CBM (CtCBM62) within the large C. thermocellum cellulosomal protein Cthe_2193 (defined as CtXyl5A), which establishes a new CBM family. Phylogenetic analysis of CBM62 members indicates that a circular permutation occurred within the family. CtCBM62 binds to d-galactose and l-arabinopyranose in either anomeric configuration. The crystal structures of CtCBM62, in complex with oligosaccharides containing α- and β-galactose residues, show that the ligand-binding site in the β-sandwich protein is located in the loops that connect the two β-sheets. Specificity is conferred through numerous interactions with the axial O4 of the target sugars, a feature that distinguishes galactose and arabinose from the other major sugars located in plant cell walls. CtCBM62 displays tighter affinity for multivalent ligands compared with molecules containing single galactose residues, which is associated with precipitation of these complex carbohydrates. These avidity effects, which confer the targeting of polysaccharides, are mediated by calcium-dependent oligomerization of the CBM.

Highlights

Enzymes that catalyze plant cell wall degradation display complex molecular architectures
CtCBM62 Is a Component of the C. thermocellum Cellulosomal Protein CtXyl5A—An initial analysis of the C. thermocellum genome was performed as described in Cantarel et al [6]
The analysis identified 72 genes encoding modular proteins with elements corresponding to CAZy families of catalytic modules and carbohydrate-binding modules (CBMs), which, based on the presence of type I dockerins, were components of the cellulosome

Summary

EXPERIMENTAL PROCEDURES

Expression, and Purification of Components of Cthe_2193—DNA encoding the following regions of CtXyl5A, GH5-CBM6-CBM13-Fn3-CtCBM62 Docl (mature CtXyl5A; see Fig. 1) and CtCBM62, were amplified using primers, containing. The amplified DNAs were cloned into NheI/XhoI-restricted pET21a, such that the encoded recombinant proteins contain a C-terminal His tag. Recombinant gene expression was as described above, as was protein purification, except that all buffers were supplemented with 10 mM ␤-mercaptoethanol. Mutagenesis—Site-directed mutagenesis was carried out using the PCR-based QuikChange method (Stratagene) deploying the primers listed in supplemental Table S1. Binding Assays—Affinity gel electrophoresis was used to screen for the binding of CtCBM62 to polysaccharides, following the method of Ref. 28. The proteins were subjected to nondenaturing PAGE, in the presence of 5 mM CaCl2, deploying parallel gels containing no ligand and the target polysaccharide at 100 ␮g/ml, respectively. The gels were stained with Coomassie Blue, and proteins that bound to the polysaccharide displayed reduced electrophoretic migration in the presence of the complex carbohydrate. The partial specific volume (v៮) of 0.7211 was calculated from the

PDB codes

RESULTS

DISCUSSION