Structural and functional analysis of truncated Fibrobacter succinogenes 1,3-1,4-β-D-glucanase mutant W203F

• Main Authors: Ching-Hua Hsiao, 蕭敬樺
• Other Authors: Li-Chu Tsai
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/8fv5uz

碩士 === 國立臺北科技大學 === 有機高分子研究所 === 97 === 1,3-1,4-β-D-glucanases hydrolyze and cleave β-1,4-glycosidic bonds precisely where β-1,3-glycosidic linkages are located prior to β-1,4-glycosidic linkages in lichenan or β-D-glucans. The truncated Fibrobacter succinogenes 1,3-1,4-β-D-glucanase (TFsβ-glucanase) in complex with its product has revealed that the β-1,3-1,4-cellotriose product spans subsites -3 to -1 in its active cleft in interaction with 14 amino acid residues. The four aromatic residues Phe40, Tyr42, Phe205, and Trp203 were found to make hydrophobic interactions with subsites -1, -2, and -3 of β-1,3-1,4-cellotriose, respectively. In order to understand the structural and functional roles of the aromatic residues, many mutants have been produced. The structure of the W203F mutant diffraction data were collected to 1.4 Å resolution. The active site topology was similar to that of the native complex structure, due to the presence in the structure of Tris molecules which occupied the place normally taken by the -1 subsite of the substrate, bound to the catalytic residues Glu56 and Glu60. In addition, two extra calcium ions were found in both mutant structures. Kinetic experiments revealed that Tris is a competitive inhibitor of the W203F mutant. This study suggests that aromatic residues in the active site of TFsβ-D-glucanase play critical roles in protein-carbohydrate binding, stabilization and catalysis.