Novel clostridial cell‐surface hemicellulose‐binding CBM3 proteins

A novel member of the family 3 carbohydrate‐binding modules (CBM3s) is encoded by a gene (Cthe_0271) in Clostridium thermocellum which is the most highly expressed gene in the bacterium during its growth on several types of biomass substrates. Surprisingly, CtCBM3‐0271 binds to at least two different types of xylan, instead of the common binding of CBM3s to cellulosic substrates. CtCBM3‐0271 was crystallized and its three‐dimensional structure was solved and refined to a resolution of 1.8 Å. In order to learn more about the role of this type of CBM3, a comparative study with its orthologue from Clostridium clariflavum (encoded by the Clocl_1192 gene) was performed, and the three‐dimensional structure of CcCBM3‐1192 was determined to 1.6 Å resolution. Carbohydrate binding by CcCBM3‐1192 was found to be similar to that by CtCBM3‐0271; both exhibited binding to xylan rather than to cellulose. Comparative structural analysis of the two CBM3s provided a clear functional correlation of structure and binding, in which the two CBM3s lack the required number of binding residues in their cellulose‐binding strips and thus lack cellulose‐binding capabilities. This is an enigma, as CtCBM3‐0271 was reported to be a highly expressed protein when the bacterium was grown on cellulose. An additional unexpected finding was that CcCBM3‐1192 does not contain the calcium ion that was considered to play a structural stabilizing role in the CBM3 family. Despite the lack of calcium, the five residues that form the calcium‐binding site are conserved. The absence of calcium results in conformational changes in two loops of the CcCBM3‐1192 structure. In this context, superposition of the non‐calcium‐binding CcCBM3‐1192 with CtCBM3‐0271 and other calcium‐binding CBM3s reveals a much broader two‐loop region in the former compared with CtCBM3‐0271. The structures of cell‐surface family 3 carbohydrate‐binding module proteins from Clostridium thermocellum (CtCBM3‐0271) and Clostridium clariflavum (CcCBM3‐1192) are reported. Both exhibited binding to xylan rather than cellulose, which is explained by them lacking the required number of binding residues in their cellulose‐binding strips. CcCBM3‐1192 does not contain the calcium ion considered to play a structural stabilizing role in the CBM3 family, leading to conformational changes in two loops of its structure.