Biochemical characterization and structural analysis of a bifunctional cellulase/xylanase from Clostridium thermocellum

Abstract

We expressed an active form of CtCel5E (a bifunctional cellulase/ xylanase from Clostridium thermocellum), performed biochemical characterization, and determined its apo- and ligandbound crystal structures. From the structures, Asn-93, His-168, His-169, Asn-208, Trp-347, and Asn-349 were shown to provide hydrogen-bonding/hydrophobic interactions with both ligands. Compared with the structures of TmCel5A, a bifunctional 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 His-168 is essential for xylanase activity, and His-169 is more important for xylanase activity, whereas Asn-93, Asn-208, Tyr-270, Trp-347, and Asn-349 are critical for both activities. In contrast, F267A improves enzyme activities.