以X光晶體結構方法來佐證醣基轉換反應：Trichoderma harzianum ETS323木聚糖[]與木聚寡糖複合物研究

以X光晶體結構方法從屬於家族11糖基水解?的木聚糖?與木聚寡糖複合物高解析度三維分子結構中得到關於木聚寡糖與酵素結合的重要資訊。由兩個木聚三糖得來的木聚六糖電子密度中顯示木聚糖?具有糖基轉機?的活性。這是第一次從蛋白質結構中看到反應物停留在家族11糖基水解?的全部六個反應單元中，發現幾個重要殘基扮演非常重要的角色。Arg122產生了方向的變異，提供了固定反應物的氫鍵，如果Arg122不產生方向上的轉變是無法提供在反應位 (-1)的氫鍵固定功能。其他接近反應區的殘基也適當提供氫鍵增加木聚寡糖反應物在反應區域的結合穩定性，而W18、Y179與Y96則利用本身具有的苯環與木糖環在反應位 (-2,+2和+3)的位置形成芳香性π軌域重疊反應，辨認適當種類的反應物進行結合。進行糖基轉換的兩個重要殘基分別是Glu86與Glu177，前者作為親核性(鹼性)端，後者則是質子提供(酸性)端，構成了連接兩個木聚寡糖間的配糖鍵。其它木聚糖?與木聚寡糖複合物的結構成果也都符合先前研究工作的發現。綜合以上的結構證據，可以解釋在酵素動力學上所得到的結果，並提供一些方向做為改良酵素活性、提高對熱忍受與設計糖?合成?的參考

關鍵字

木聚糖[] ；反應物結合；木聚寡糖；複合物結構；糖?合成?

並列摘要

The high resolution (1.2-1.4 ?) complex three-dimensional structures of xylanase (family 11) provide the xylooligosaccharide substrates binding information by X-ray crystallography. The electron density of xylohexaose from two xylotriose show GH-11 xylanase have glycosyltransferase activity in the xylotriose soaking data. It is the first time to capture that the 6 subsites abound with substrates. Arg122 changed the orientation to offer a hydrogen bond to fix xylooligosaccharides in the catalytic site. Other residues were close to the catalytic pocket also form hydrogen bonds to stabilize the substrates. W18 , Y179 and Y96 interact with the xylopyranose rings by aromatic stacking at the subsite (-2,+2 and +3) to recognize substrate binding actions. Two glutamic acids, Glu86 and Glu177 are a nucleophile (base) and a proton donor (acid), interact with substrates at subsite (-1) to build up a glycosidic bond to connect two xylotriose sugars. Other complex structure datas confirm previous reports. The structural information can explain the GH-11 xylanase kinetic data and offer a direction to improve the specify activity, heat tolerant and glycosynthase activity.

並列關鍵字

Xylanase ； substrate binding ； xylooligosaccharide ； complex structure ； glycosynthase

參考文獻

[1] Collins T, Gerday C & Feller G. Xylanases, xylanase families and extremophilic xylanases. Fems Microbiology Reviews (2005) 29: pp. 3-23.

[2] Kulkarni N, Shendye A & Rao M. Molecular and biotechnological aspects of xylanases. Fems Microbiology Reviews (1999) 23: pp. 411-456.

[4] Henrissat B & Davies G. Structural and sequence-based classification of glycoside hydrolases.. Curr Opin Struct Biol (1997) 7: pp. 637-644.

[5] T?rr?nen A & Rouvinen J. Structural comparison of two major endo-1,4-xylanases from trichoderma reesei.. Biochemistry (1995) 34: pp. 847-856.

[6] Schmidt A, Gubitz GM & Kratky C. Xylan binding subsite mapping in the xylanase from penicillium simplicissimum using xylooligosaccharides as cryo-protectant. Biochemistry (1999) 38: pp. 2403-2412.

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以X光晶體結構方法來佐證醣基轉換反應：Trichoderma harzianum ETS323木聚糖[]與木聚寡糖複合物研究

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