Biochemical characterization and structural analysis of a bi-functional cellulase/xylanase from Clostridium thermocellum [Enzymology]

January 9th, 2015 by Yuan, S.-F., Wu, T.-H., Lee, H.-L., Hsieh, H.-Y., Lin, W.-L., Yang, B., Chang, C.-K., Li, Q., Gao, J., Huang, C.-H., Ho, M.-C., Guo, R.-T., Liang, P.-H.

We expressed an active form of CtCel5E (a bifunctional cellulase/xylanase from Clostridium thermocellum), performed biochemical characterization, and determinedsolved its apo and ligand-bound crystal structures. From the structures, Asn93, His168, His169, Asn208, Trp347, and Asn349 were shown to provide H-bonding/hydrophobic interactions with both ligands. Compared with to the structures of TmCel5A, a bi-functional cellulase/mannanase homolog from Thermotoga maritima, a flexible loop region in CtCel5E is the key for discriminating substrates. Moreover, site-directed mutagenesis data confirmed that His168 is essential for xylanase activity, His169 is more important for xylanasecellulase activity, Asn349 is only required for cellulase activity, whereas Asn93, Asn208, Tyr270, and Trp347 and Asn349 are critical for both activities. In contrast, F267A improves enzyme activities.