米根黴菌葡糖澱粉?之澱粉結合蛋白與β環形糊精及麥芽七糖複合物晶體結構之研究

Crystal Structures of Starch Binding Domain from Rhizopus oryzae Glucoamylase in Complex with β-Cyclodextrin and Maltoheptaose

劉儼毅(Yen-Yi Liu)

指導教授 ： 孫玉珠

國立清華大學/生命科學院/生物資訊與結構生物研究所/碩士(2008年)

摘要

葡糖澱粉?(glucoamylase)是一種外切水解?，作用在澱粉非還原端及一些寡醣上的α-1,4 與α-1,6 鍵結，水解澱粉及多醣類變成葡萄糖，米根黴菌葡糖澱粉?(RoGA)包含有兩個功能蛋白(domain)，一為N端的澱粉結合蛋白 (Starch-binding domain);另一為C端的水解催化蛋白(catalytic domain)，這二個蛋白以O型配醣體連結區域(o-glycosylated linker)連接。葡糖澱粉?的澱粉結合蛋白屬於醣類結合模組第二十一族(CBM21)。我們利用X光晶體繞射實驗決定澱粉結合蛋白與β環形糊精及麥芽七糖之晶體結構，所得到晶體解析度分別達1.8 ?及2.3 ?，整個澱粉結合蛋白的結構屬於β三明治摺疊(β-sandwich)且具有類似免疫球蛋白(immunoglobulin-like)的結構。米根黴菌葡糖澱粉?中的澱粉結合蛋白具有兩個醣類結合位置(carbohydrate-binding site)：結合位置I主要由色胺酸47、酪胺酸83及酪胺酸94等三個保守的芳香族殘基組成，這幾個胺基酸殘基形成一個寬平且堅實的疏水結合表面。結合位置II主要由酪胺酸32及苯丙胺酸58組成，這兩個胺基酸殘基則是形成一個突出狹長的結合環境。此外，一些親水性胺基酸殘基，例如位於結合位置I的天門冬醯酸50、96、101及結合位置II的天門冬醯酸29、麩胺酸68、離胺酸34，也都有參與糖的結合。 澱粉結合蛋白在與醣類結合時，其構型與未結合醣類時的蛋白構型差異不大，主要的不同處是在環套區以及醣類結合位置附近，其中結合位置I的結構差異性又比結合位置II來的大。另外，當我們比較了黑麴菌與米根黴菌葡糖澱粉?中的澱粉結合蛋白與β環形糊精的複合結構時，發現到，雖然二者結構中的β環形糊精黏附蛋白結構的形式類似，但是二者結構中的β環形糊精在空間上的方位有差異，這個差異可能使得這兩種澱粉結合蛋白與澱粉有不同的結合模式。除此之外，在表面電位分佈上(electrostatic potential surface)上可以觀察到有一明顯的疏水表面，而其背面則呈現正負電分開分佈，由晶體的堆疊中可以發現相鄰的蛋白分子互相以正負電互補的方式排列。由此可推測，當一個澱粉結合蛋白以疏水面結合於澱粉顆粒表面時，可透過其背側的帶電表面吸引其他澱粉結合蛋白靠近澱粉顆粒表面。從澱粉結合蛋白的表面電位分佈以及那些參與醣類結合的胺基酸殘基,可以推測澱粉結合蛋白在參與葡糖澱粉?水解澱粉時所扮演的角色。

關鍵字

米根黴菌葡糖澱粉? ； 澱粉結合蛋白 ； β環形糊精 ； 麥芽七糖 ； 晶體結構

並列摘要

Glucoamylase hydrolyzes starch and polysaccharides to β-d-glucose. Rhizopus oryzae glucoamylase (RoGA) consists of two functional domains, an N-terminal starch binding domain (SBD) and a C-terminal catalytic domain and these two domains are connected by an O-glycosylated linker. The starch-binding domain of RoGA belongs to carbohydrate-binding module 21(CBM21). Two crystal structures of the RoGACBM21 complexes, with a cyclic carbohydrate, β-cyclodextrin (RoGACBM21-βCD), and with a linear carbohydrate, maltoheptaose (RoGACBM21-G7), were determined at 1.8 and 2.3? respectively. The overall structures belong to a β-sandwich fold with an immunoglobulin-like structure. Two carbohydrate-binding sites were observed. Site I is created by several conserved aromatic residues, Trp47, Tyr83, and Tyr94, which form a broad, flat, and firm hydrophobic binding surface. Site II is built up by Tyr32 and Phe58, which produce a protruded and narrow binding environment. Besides, some hydrophilic residues which are Asn50, Asn96, Asn101 in Site I and Asn29, Glu68, Lys34 in Site II also participate in carbohydrates binding. Liganded and unliganded RoGACBM21s reveal similar overall structures, the major structural difference was found in loop regions and in or near the two carbohydrate-binding sites. Site I undergoes a bigger conformational change than that in site II upon the carbohydrate binding. The overall structure and ligand binding mode of AnGACBM20-βCD complex is similar to RoGACBM21-βCD but the orientations of the βCD at site I and II are different. As for electrostatic potential of RoGACBM21-βCD, it appears a more hydrophobic surface than other surfaces throughout the structure and its opposite side has positive and negative charges separately. Therefore, we can make a suggestion that once a SBD molecule binds to the starch granule surface, it may attract more SBD molecules onto the surface through electrostatic complementation. In conclusion, from electrostatic potential of RoGACBM21-βCD and the ligand binding residues, we could propose a possible role of SBD participating in glucoamylase hydrolysis on the starch granular surface.

並列關鍵字

RoGACBM21 ； SBD ； RoGACBM21-βCD ； RoGACBM21-G7 ； βCD ； maltoheptaose ； crystal structure

參考文獻

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