標題: 醣苷水解酵素家族29與64的反應機制與重要催化殘基之鑑定
Catalytic mechanistic and identification of essential residues of Glycohydrolase in family GH-29 and GH-64
作者: 柳勝文
Liu, Sheng-Wen
李耀坤
Li, Yaw-Kuen
應用化學系碩博士班
關鍵字: 醣苷水解酵素;α-L-岩藻糖水解醣苷酵素;β-1,3-葡聚五糖生產水解醣苷酵素;反應機制;重要催化殘基;保留構型機制;反轉構型機制;Glycohydrolase;α-L-Fucosidase;Laminaripentaose-producing β-1,3-glucanase;Catalytic mechanistic;essential residues;retaining mechanistic;inverting mechanistic
公開日期: 2008
摘要: 摘要: 第一部分:家族29 之人類α-L-岩藻糖水解醣苷酵素的蛋白質表現、突變以及催化反應機制和重要殘基的研究與探討 人類α-L-岩藻糖水解醣苷酵素(Human α-L-fucosidase,h-Fuc)屬於醣苷類水解酵素第29家族的水解酵素(EC 3.2.1.51),本研究著重於該酵素的相關催化功能、反應機構與催化重要殘基之鑑定探討。本研究成功地將人類肝細胞組織α-L-岩藻糖水解醣苷酵素表現於大腸桿菌系統中。首先,將人類肝細胞組織α-L-岩藻糖水解醣苷酵素之基因,重組建構在pET22b(+)表現質體上,後轉殖進入大腸桿菌BL21(DE3)系統中表現,於pH值6.0的LBA液體培養基中,並以IPTG誘導培養;後經由離子交換管柱(SP、Q)及G-75凝膠管柱等層析管柱純化處理,可以取得均質度達95%的重組酵素,酵素單體的分子量約為50 kDa左右。以p-nitrophenyl-α-L-fucopyranoside (pNPF)為反應受質進行對重組h-Fuc酵素之催化活性分析。實驗結果顯示重組h-Fuc酵素在pH 5.0的環境下,求得其Km以及kcat分別為0.105 mM和48.6 s-1,相較於文獻中自人類肝臟萃取純化之原生h-Fuc酵素(Km = 0.43 mM,kcat = 16.3 sec-1),重組之h-Fuc酵素的催化能力(kcat/Km)約為肝臟原生酵素的12倍。同時,亦得知在溶液 pH 4.5以及 pH 6.5左右存在有兩活性最佳區域,而當溶液pH值小於3.0或是大於7.5後,酵素即會呈現不穩定狀態,使催化活性下降。利用1H-NMR光譜分別觀測h-Fuc酵素催化水解受質pNPF的立體選擇性,與在含有特定比例甲醇/水之溶液中,進行h-Fuc酵素的轉醣反應,發現在以不同芳香族離去基之糖苷受質與h-Fuc酵素作用反應下,催化過程皆經由一fucosyl-enzyme中間體的形成,得知其h-Fuc酵素催化反應為一似SN1的雙步驟取代之保留構型機制。並利用化學合成法合成一系列具有不同芳香族離去基之糖苷受質進行Brǿnsted relationship之研究,得到Brǿnsted constant(log kcat之βlg值為-0.13與log kcat/Km之βlg值為-0.27),推測酵素催化速率決定步驟應為醣基化(fucosylation)步驟。隨後我們藉由已知的Tm-Fuc酵素晶體結構,進行h-Fuc酵素的蛋白結構與胺基酸二級結構序列之模擬分析,選擇以位於β-平板結構上的八個高保留殘基,進行酵素之定點突變,觀測各突變株酵素與受質pNPF的動力學反應速率分析。發現有兩胺基酸D225G和E289G之突變株酵素在反應速率kcat/Km值,分別降為野生株的20,000和450倍以上。接著在不同pH值緩衝溶液下,以此兩定點突變株酵素進行pH-profile曲線的反應活性表現,結果顯示出兩胺基酸的突變株酵素與野生株有著很大差異。並利用加入親核性陰離子試劑對兩突變株D225G和E289G酵素進行化學活性復活技術。當以外加疊氮陰離子分別對D225G和E289G兩突變株進行化學活性復活反應,發現催化反應速率kcat值分別可增加約6和24倍,並於氫核磁共振磁譜分析出β-fucosyl_azide與α-fucosyl_azide的兩突變酵素作用產物。最後,以突變株E289G酵素與受質pNPF反應一段時間後,於液相質譜中偵測到岩藻糖-酵素之過渡狀態中間體的累積產物分子量。這些實驗結果指出h-Fuc酵素催化反應中扮演親核基和一般酸鹼基分別為Asp225 和Glu289。這是首次完成人類α-L-岩藻醣苷水解酵素之反應機構與重要催化殘基鑑定的研究。 第二部分:家族64的鏈黴菌DIC-108之β-1,3-葡聚五糖生產水解糖苷酵素的人造基因選殖、蛋白質表現與突變以及蛋白質結構和重要殘基的研究與探討 本研究旨在探討自Streptomyces matensis DIC-108 菌株之β-1,3-葡聚五糖生產水解糖苷酵素(Laminaripentaose-producing β-1,3-glucanase, LPHase)屬於醣苷水解酵素第64家族的水解酵素(EC 3.2.1.39),藉以單取代(single-displacement)反轉機制催化水解β-1,3糖苷鍵結,並專一性地生產由五個葡萄糖所構成的laminaripentaose寡糖產物。將著重於該酵素的性質、催化功能、反應機構與催化重要殘基的鑑定,並進一步對此酵素的蛋白質晶體結構作解析與探討。本研究首先,以24條人造設計之寡核苷酸引子,利用PCR技術,合成完整的LPHase人造基因,並重組建構於pRSET_A表現載體上,後轉殖進入大腸桿菌BL21(DE3)系統中,以IPTG誘導過量表現。經過各離子交換層析管柱(SP、Q、CM)等純化處理,可以取得均質度達95%的酵素,酵素單體的分子量約為40 kDa左右。以自製膠狀卡德蘭膠(curdlan)為反應受質進行活性分析,實驗結果顯示重組之LPHase酵素在溫度55 0C及pH 7.5~8.5為活性最佳區域。當溶液pH值小於3.5或是大於11.0後,酵素會呈現不穩定狀態。利用1H-NMR觀測LPHase酵素催化水解受質糖的立體選擇性作用,為一似SN2之單取代反轉構型機制。隨後我們以純化之LPHase酵素進行卡德蘭膠聚糖之水解反應,利用透析膜的孔洞大小來篩選生產高純度laminaripentaose寡糖產物;並以化學合成具有芳香族離去基團之糖苷受質進行酵素催化反應速率之動力學研究。將合成之p-NLPG糖苷受質在40 0C,50 mM磷酸緩衝溶液(pH 7.1)的反應環境下,與LPHase酵素進行催化水解作用,其所得Km以及kcat分別為1.6 mM和8.1 s-1。當LPHase酵素在各不同酸鹼值緩衝溶液中,其反應活性之pH-profile呈現一對稱鐘型曲線(bell-shaped curve),由曲線得知調控LPHase酵素所扮演的兩個重要催化殘基之解離值pKa1和pKa2分別為6.0及10.1。隨後,將糖苷水解酵素家族GH-64中,所有子家族來源菌的胺基酸序列進行多重序列比對分析,發現了五個完全高保留度位置的胺基酸,分別為Asp143、Glu154、Asp170、Asp376及Asp377。結合酵素之定點突變學,觀測各突變株酵素與受質卡德蘭膠水解的相對催化活性,並量測對合成之糖苷受質p-NLPG的反應初始速率變化;發現有兩突變株E154G和D170G酵素的反應速率kcat/Km值,降低約為野生株的1,700~2,000倍。接著以利用加入親核性陰離子試劑對兩突變株E154G和D170G酵素進行化學活性復活技術,結果得知突變株D170G酵素的催化反應活性,可被加入的疊氮陰離子所復活並於液相質譜與氫核磁共振磁譜分析α-laminaripentaosyl_azide產物的產生。鑑定出LPHase酵素在催化反應過程中,扮演一般鹼催化基團(親核性基)為Asp170和一般酸催化基團為Glu154。並配合LPHase酵素突變學與蛋白晶體結構,得知胺基酸Arg115,為影響此酵素在高pH值溶液的催化作用;和胺基酸Tyr232,輔助對扮演一般鹼催化基團附近水分子之氫鍵鍵結作用力有相當重要性。利用X-ray晶體繞射方式取得LPHase酵素完整晶體結構。分別收集來自各不同結晶長成之反應條件母液中,原始酵素與硒化甲硫胺酸酵素(SeMet-LPHase)的蛋白質晶體,利用多波長非尋常散射(MAD)的方式,收取解析度達1.62~2.3 Å 的晶體繞射數據。鑑定出LPHase酵素晶體完整架構,是有6個α-螺旋結構、18個β-平板結構和1個η結構,以上下交錯之β-平板結構的β-桶狀區域及α/β 混合區域所建構而成,為一大開口裂縫(open groove)內切型外觀結構之糖苷水解酵素。由蛋白複合體晶體的結構模子,判定在酵素催化中心含至少五個糖苷分子以上鍵結的受質結合次單位區域。自聚糖鏈還原端向非還原端一方,由扮演一般鹼催化Asp170和一般酸催化Glu154的兩重要殘基,以似SN2之單取代反轉催化機制的外切型態進行水解反應,形成以β-1,3鍵結而成的專一laminaripentaose寡醣產物。兩催化殘基之胺基酸立體空間距離約為8 ± 0.5 Å。
ABSTRACT Part I : Expression, mutagensis, mechanistic study and identification of essential residues of human α-L-Fucosidase in family GH-29 Fucosylated glycoconjugates play critical roles in various biological processes, but the limited availability of α-L-Fucosidase hampers investigations at a molecular level. This thesis aims to clone and express human Fucosidase. A gene from human encoding α-L-fucosidase (Fuc) was cloned into pET22b(+) plasmid. Protein was successfully expressed in E. coli BL21 (DE3). After applying a series of ion-exchange and gel-filtration chromatography purification steps, recombinant h-Fuc with 95% homogeneity can be obtained. The molecular weight of the enzyme was analyzed by SDS-PAGE to be about 50 kDa. The optimal temperature of h-Fuc was 70 0C and the optimal pH was 4.5 and 6.5. The h-Fuc was stable at pH 3.0~6.0, while it was unstable at 75 0C or high. The catalytic mechanism of h-Fuc was confirmed a SN1-like with two-step double displacement of retaining enzyme by transglycosylation and 1H-NMR spectroscopy. Based on careful sequence alignment of GH-29 enzymes and extensive structure analysis of the close homologues of h-Fuc, nine residues of glutamate and aspartate in h-Fuc were selected for mutagenic study to determine the essential residues. Among the mutants, D225N, E289Q and E289G lost catalytic activity significantly; their kcat values are 1/5,700, 1/430 and 1/340, respectively, of that of the wild-type enzyme. Based on kcat values of aryl-α-L-fucopyranosides catalyzed by wild-type α-L-fucosidase, a small Brønsted constant, βlg = −0.13, was derived, indicating that the rate-limiting step of the enzymatic reaction is fucosylation. The Brønsted plot for kcat/Km for the E289G mutant is linear with βlg = −0.93, but that for kcat is biphasic, with βlg for poor substrates being −0.88 and for activated substrates being −0.11. The small magnitude of βlg for the activated substrates may indicate that the rate-limiting step of the reaction is defucosylation, whereas the large magnitude of the latter βlg value for the poor substrates indicates that the rate-limiting step of the reaction becomes fucosylation. The kinetic outcomes support an argument that Asp225 functions as a nucleophile and Glu289 as a general acid/base catalyst. As further evidence, azide significantly reactivated D225G and E289G, and 1H-NMR spectral analysis confirmed the formation of β-fucosyl azide and α-fucosyl azide in the azide rescues of D225G and E289G catalyses, respectively. As direct evidence to prove the function of Glu289, an accumulation of fucosyl-enzyme intermediate was detected directly through ESI/MS analysis. Part II:Artificial gene cloning、protein mutagensis and crystal structure study and identification of essential residues of LPHase from Streptomyces matensis DIC-108 in family GH-64 An artificial gene of laminaripentaose-producing β-1,3-glucanase (LPHase) from Streptomyces matensis DIC-108 was reconstructed by PCR and overexpressed in E. coli. Although the polypeptide sequence of LPHase exhibits significant similarity with the catalytic domains of β-1,3-glucanase of Arthrobacter sp. YCWD3 and Cellulosimicrobium cellulans, it uniquely catalyzes the hydrolysis of β-1,3-glucans to release laminaripentasaccharide as the predominant product. The recombinant wild-type enzyme and mutants were purified to >90% homogeneity by ionic-exchange chromatography. The optimal temperature of the LPHase was 55 oC and the optimal pH was 8.5. The LPHase was stable at pH 5.0~9.0, while it was unstable at 65 0C or higher. The catalysis of LPHase is confirmed to follow a one-step single displacement mechanism by 1H-NMR spectroscopy. p-Nitrophenyl-β-1,3-laminaripentaopyranoside (p-NPLPG) was synthesized to facilitate the kinetic analysis. Michaelis constant (Km) and catalytic activity were determined with p-NPLPG and were found to be 1.6 mM and 8.1 s-1, respective. To determine the amino acid residues essential for the catalysis of LPHase, more than 10 residues including five highly conserved residues, D143, E154, D170, D376 and D377, were mutated. Among the mutants, E154 and D170 mutants, such as E154Q, E154G, D174N, and D174G, significantly lost their catalytic activity. Further investigation with chemical rescue on E154G and D174G confirmed that E154 and D170 function as the general acid and the general base in the LPHase catalysis, respectively. This study provided the first hand information on identification of the essential residues GH-64 through kinetic examination. Further studies on structural and enzymatic characterizations were also conducted to understand the detail catalytic mechanism of LPHase. The crystal structure of LPHase was solved to 1.62 Å resolution using multiple-wavelength anomalous dispersion methods. The LPHase structure reveals a novel crescent-like fold; it consists of a barrel domain and a mixed (□□□) domain, including six □-helix, eighteen □-sheet, and one □ structure, forming a wide-open groove between the two domains. The liganded crystal structure was also solved to 1.80 Å, showing limited conformational changes. Within the wide groove, molecular modeling using a laminarihexaose as a substrate suggests roles for Glu154 and Asp170 as acid and base catalysts, respectively, whereas the side chains in active site demarcate subsite +5. Site-directed mutagenesis of Glu154 and Asp170 confirms that both carboxylates are essential for catalysis. Together, our results suggest that LPHase uses a direct displacement mechanism involving Glu154 and Asp170 to cleave a □-1,3-glucan into specific α-pentasaccharide oligomers.
URI: http://140.113.39.130/cdrfb3/record/nctu/#GT009025523
http://hdl.handle.net/11536/38047
Appears in Collections:Thesis


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