Glycoside hydrolase (GH) family 5 is one of the largest GH families with various GH activities 33 including lichenase, but the structural basis of the GH5 lichenase activity is still unknown. A novel 34 thermostable lichenase F32EG5 belonging to GH5 was identified from an extremely thermophilic 35 bacterium Caldicellulosiruptor sp. F32. F32EG5 is a bi-functional cellulose and lichenan-degrading 36 enzyme and exhibited a high activity on β-1,3-1,4-glucan but side activity on cellulose. Thin-layer 37 chromatography and NMR analyses indicated that F32EG5 cleaved the β-1,4 linkage or the β-1,3 38 linkage while a 4-O-substitued glucose residue linked to a glucose residue through a β-1,3 linkage, 39 which is comp... More
Glycoside hydrolase (GH) family 5 is one of the largest GH families with various GH activities 33 including lichenase, but the structural basis of the GH5 lichenase activity is still unknown. A novel 34 thermostable lichenase F32EG5 belonging to GH5 was identified from an extremely thermophilic 35 bacterium Caldicellulosiruptor sp. F32. F32EG5 is a bi-functional cellulose and lichenan-degrading 36 enzyme and exhibited a high activity on β-1,3-1,4-glucan but side activity on cellulose. Thin-layer 37 chromatography and NMR analyses indicated that F32EG5 cleaved the β-1,4 linkage or the β-1,3 38 linkage while a 4-O-substitued glucose residue linked to a glucose residue through a β-1,3 linkage, 39 which is completely different from extensively studied GH16 lichenase that catalyses strict 40 endo-hydrolysis of the β-1,4-glycosidic linkage adjacent to a 3-O-substitued glucose residue in the 41 mixed linked β-glucans. The crystal structure of F32EG5 was determined to 2.8 Å resolution and 42 the crystal structure of the complex of F32EG5 E193Q mutant and cellotetraose was determined to 43 1.7 Å resolution, which revealed that the exit subsites of substrate binding sites contribute to both 44 thermostability and substrate specificity of F32EG5. The sugar chain showed a sharp bend in the 45 complex structure, suggesting that a substrate cleft fitting to the bent sugar chains in lichenan is a 46 common feature of GH5 lichenases. The mechanism of thermostability and substrate selectivity of 47 F32EG5 was further demonstrated by molecular dynamics simulation and site-directed 48 mutagenesis. These results provide biochemical and structural insight into thermostability and 49 substrate selectivity of GH5 lichenases which have potential in industrial processes.
