Abstract
Glycoside hydrolase family 12 endocellulase (GH family12) plays a key role in the degradation of β-glucan and cellulose. Hyperthermostable GH family 12 endocellulase from the archaeon Pyrococcus furiosus (EGPf) catalyzes the hydrolysis of β(1 → 4) glucosidic linkages in cellulose and β-glucan containing β(1 → 3),(1 → 4) mixed-linkages. Therefore, in the combination with the hyperthermophilic β-glucosidase from P. furiosus (BGLPf), non-crystalline cellulose and β-glucan can be degraded to glucose completely by EGPf at high temperature. X-ray crystallography and protein engineering were used to reveal how the β(1 → 4) and β(1 → 3) linkages in β-glucan substrates are recognized by the enzyme. Structural and functional analyses clarified that the active site of EGPf consists of six subsites: the reducing end subsites (+1 and + 2) recognize both β(1 → 4) and β(1 → 3) linkages of various substrates in a productive binding mode, and recognition is controlled by Trp121 and Gln208 located at subsite +2. It was also revealed that the deep cleft in subsite −4 can accommodate the torsion angles of substrates consisting of β(1 → 3),(1 → 4) mixed-linkages due to the changing tilt of the Trp62 side chain. From the structural similarity, it is proposed that the substrate specificity of family 12 endocellulases towards β(1 → 3),(1 → 4) mixed-linkage substrates are controlled by the subsites (+1, +2, and −4). Furthermore, the function of family 12 endocellulase could be improved by protein engineering method using the information of the analysis.
Published Version
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