截短型Fibrobacter succinogenes 1,3-1,4-β-D-葡聚醣水解酶突變種W203F結構及功能分析

1,3-1,4-β-D-葡聚醣水解酶能專一性催化水解β-1,3-1,4-葡聚醣或地衣聚醣中連結於β-1,3鍵結後之β-1,4醣苷鍵。從截短型Fibrobacter succinogenes 1,3-1,4-β-D-葡聚醣水解酶產物β-1,3-1,4-纖維三醣複合體結構中得知三醣位於在其活性裂縫凹槽之-1到-3之範圍並和14個胺基酸有鍵結。其中芳香族胺基酸Phe40, Tyr42, Phe205, and Trp203分別可以與b-1,3-1,4纖維三醣之-1,-2,和-3醣單元產生疏水性作用力。為了進一步瞭解這些芳香族氨基酸與功能之間的相關聯性，許多突變種酵素已經被構築完成。我們獲得突變種W203F的蛋白質晶體並收集到繞射數據解析度為1.4 Å。突變種W203F晶體結構中發現Tris分子佔據在原本催化活性區域-1醣單元結合位置並和主要扮演催化水解氨基酸Glu56及Glu60有氫鍵鍵結，可能導致突變種W203F催化活性區結構形態跟原生種纖維三醣複合體的結構非常相似。動力學分析實驗數據證明Tris分子對W203F扮演競爭型抑制劑的角色。由晶體結構和動力學數據得知位於1,3-1,4-β-D-葡聚醣水解酶活性區域內之芳香族胺基酸，在蛋白質及受質碳水化合物的結合、穩定和催化上扮重要的角色。

關鍵字

醣苷水解酶芳香族胺基酸；疏水性作用力；三羥甲基氨基甲烷；競爭型抑制劑

並列摘要

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.

並列關鍵字

glycosyl hydrolase ； aromatic residues ； hydrophobic interactions ； tris(hydroxymethyl)aminomethane ； competitive inhibition

參考文獻

1. Hahn, M., J. Pons, A. Planas, E. Querol, et al., Crystal structure of Bacillus licheniformis 1,3-1,4-β-D-glucan 4-glucanohydrolase at 1.8Å resolution. FEBS Letters, 1995. 374(2): p. 221-224.

2. Henrissat, B., A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochemical Journal, 1991. 280(2): p. 309-316.

3. Henrissat, B. and A. Bairoch, New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochemical Journal, 1993. 293(3): p. 781-788.

4. Malet, C., J. Vallé, J. Bou, and A. Planas, A specific chromophoric substrate for activity assays of 1,3-1,4-b-glucan 4-glucanohydrolases. Journal of Biotechnology, 1996. 48(3): p. 209-219.

5. Withers, S.G., Mechanisms of glycosyl transferases and hydrolases. Carbohydrate Polymers, 2001. 44(4): p. 325-337.

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截短型Fibrobacter succinogenes 1,3-1,4-β-D-葡聚醣水解酶突變種W203F結構及功能分析

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