澱粉吸附區與醣類交互作用之模型

An Interaction Model between Glycans and Starch Binding Domain of Rhizopus oryzae Glucoamylase

黃瀚賜(Han-Tze Huang)

指導教授 ： 張大慈

國立清華大學/生命科學院/分子與細胞生物研究所/碩士(2012年)

https://doi.org/10.6843/NTHU.2012.00119

摘要

米根黴菌 (Rhizopus oryzae) 葡萄糖澱粉酵素 (glucoamylase) 為579個胺基酸組成的醣類水解酶，其整體結構包含胺基端澱粉吸附區 (starch-binding domain, RoSBD)、羧基端酵素催化區及一段高度醣基化的連接片段。RoSBD隸屬於醣類吸附模組 (carbohydrate-binding module, CBM) 家族二十一，對天然澱粉與可溶性多醣具有強結合力。核磁共振與晶體繞射解析證明RoSBD具有兩個配體結合位：第一個結合位由色胺酸47、酪胺酸83、酪胺酸93、及酪胺酸94組成，第二個結合位由酪胺酸32、苯丙胺酸58組成；兩個RoSBD各提供一個不同的配體結合位與單一醣分子結合。一般而言，串接的醣類吸附模組對醣類的結合力較強；因此本研究設計以一段短胜肽鏈串接兩個RoSBD，並探討其對醣類的結合力值變化。利用恆溫滴定熱量計 (isothermal titration calorimetry, ITC) 測量雙聚體RoSBD與多種不同醣類的結合力。ITC實驗結果顯示雙聚體RoSBD對多種不同醣類的結合力都高於單體RoSBD。除此之外，本研究亦將RoSBD的主要配體結合胺基酸突變成丙胺酸，根據測量突變蛋白質對不同醣類之結合力變化配合RoSBD之三級結構，可預測雙聚體RoSBD和不同醣類之結合模式。對於短鏈或環形醣類而言，以結晶繞射解析的結果去分析，雙聚體中兩個RoSBD分子各提供一個不同的配體結合位與醣分子結合。若以恆溫滴定熱量計的數據去分析，在雙聚體RoSBD之N端的酪胺酸32對於短鏈醣類的結合是很重要的，然而在雙聚體RoSBD之C端的酪胺酸32對於環形醣的結合是很重要的;而對於長鍊醣類而言，酪胺酸32扮演著極為重要的角色，推測雙聚體中兩個RoSBD分子的皆以酪胺酸32與長鏈醣分子結合，兩個RoSBD分子形成面對面之模型。本研究發現RoSBD會和不同種類的醣類有不同的結合模型。了解RoSBD和醣類的結合模式，能更深入探討醣類吸附模組與不同醣類結合的分子機制。

關鍵字

葡萄糖澱粉酵素 ； 澱粉吸附區 ； 醣類吸附模組

並列摘要

The N-terminal starch binding domain of Rhizopus oryzae glucoamylase (RoSBD) belongs to carbohydrate binding module (CBM) family 21 with high binding affinities toward raw starches, and contains two ligand binding sites at opposite position (Site I: Trp47, Tyr83 and Tyr94; Site II: Tyr32, Phe58). Three dimensional structure reveals that two RoSBD molecules hold the same sugar ligand by different binding sites. Natural CBMs in tandem repeat show higher ligand-binding affinity than single CBM does, yet whether two cooperative RoSBDs enhance ligand binding properties remains to be investigated. In this study, dimeric RoSBD was constructed with a short peptide linker between two RoSBD units. Isothermal titration calorimetry (ITC) data indicated that dimeric RoSBD processed higher affinities toward a series of glycans than monomeric RoSBD. Moreover, site-directed mutagenesis of major ligand binding residues Y32 and W47 on each domain of dimeric RoSBD revealed determining factors by ITC analysis. In addition, in silico structure modeling was carried out by quantitative measurement of binding affinity between wild-type/mutant dimeric RoSBD and soluble starch by ITC and published X-ray crystallography data. For short glycans such as maltoheptaose (Glc7), or cyclic ligand β-cyclodextrin (βCD), two RoSBD molecules might hold on the same sugar ligand by different binding sites based on X-ray crystallography data to predict. However, according to ITC data, Y32 on the N-terminal RoSBD played a crucial role in Glc7 binding and Y32 on the C-terminal RoSBD was important for βCD binding. For long chain glycans, Y32 in both two RoSBD molecules played a crucial role in ligand binding, indicated that two binding site II of dimeric RoSBD bound to a same glycan to form RoSBD/RoSBD interface. In conclusion, RoSBD might bind to various glycans in different interaction models. The interaction model between RoSBD and glycans will greatly increase our understanding in the molecular mechanisms of CBM functions.

並列關鍵字

glucoamylase ； starch binding domain ； carbohydrate binding module

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