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研究生:王詠霖
研究生(外文):Yung-Lin Wang
論文名稱:聚三羥丁酸降解酶及海藻糖合成酶:酵素結構、催化機制與蛋白工程
論文名稱(外文):Poly-3-hydroxybutyrate depolymerase and trehalose synthase:Structure, Function and Engineering
指導教授:廖淑惠廖淑惠引用關係
指導教授(外文):Shwu-Huey Liaw
學位類別:博士
校院名稱:國立陽明大學
系所名稱:生化暨分子生物研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:93
中文關鍵詞:聚三羥丁酸降解酶海藻糖合成酶酵素結構
外文關鍵詞:poly-3-hydroxybutyrate depolymerasetrehalose synthasestructures
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我的博士論文是研究蘇力氏菌 (Bacillus thuringiensis) 的聚三羥丁酸降解酶(BtPhaZ)與抗輻射奇異球菌 (Deinococcus radiodurans)的海藻糖合成酶 (DrTS),這兩個酵素分別可應用在生物塑膠的分解及抗生素原料之工業製造,與高經濟價值海藻糖的大量生產。首先我利用X光繞射法測定酵素晶體結構,並與其相似結構比對分析,進而設計突變株來研究酵素催化機制與受質專一性,希望能有助於蛋白工程開發,得到高活性、高穩定度或更高價值產物的突變株,以利於工業或醫藥上運用。
BtPhaZ催化聚三羥丁酸的降解而形成三羥丁酸 (3HB),其蛋白晶體是由本實驗室陳加林所培養,並收集1.42 Å解析度的X光繞射數據。因沒有合適的硒化甲硫胺酸BtPhaZ的晶體,因此我嘗試使用分子取代法來測定結構,雖然與已知的蛋白結構序列相同度都低於25%,再使用20個不同模板後,幸運地成功測定第一個胞內聚三羥丁酸降解酶之晶體結構。BtPhaZ含有一個典型/ hydrolase fold與一個helical domain,與相似結構比對分析,除了GxS102xG是/水解酶超家族中最高度保留區外,還發現三個新保留區,分別為HG36、D61xxGxG和G248xxD,因此設計了G36A、D61A和G248A突變株。活性分析實驗顯示G36A與 G248A分別只具有野生株活性的5%與23%,且G36A kcat較野生株下降超過1萬倍,可能是因為Ala36和Ala248分別會與Trp101與Cys277太接近,而造成活性中心結構有些改變,致使活性下降。另外D61A則表現在內涵體中 (inclusion body),因此Asp61與Ser40、Ser41和Asn67之間的交互作用,對結構的穩定性扮演重要的功能。這四個新保留區不僅組成催化三元體和oxyanion hole,而且彼此間形成的交互作用可維持活化態構型。BtPhaZ與其相似結構的酵素都具有類似的活性中心,因此可建立 BtPhaZ-3HB trimer的模擬複合結構,顯示參與受質結合之可能胺基酸,因此設計N37A、N37D、T39V、Y133F、Y133A、N214A和N214D突變株。活性分析的顯示,T39V、Y133F、Y133A、N214A和N214D與野生株有相似的活性,然而N37D和N37A只有野生株活性的20%,且N37D隨著活性分析溶液pH值上升而活性下降,在pH 9時甚至無法偵測到酵素活性,但N37D並不影響kcat,由BtPhaZ-3HB trimer模擬結構推測Asn37N會與3HB位於+2 stie上的羰基(carbonyl group)產生交互作用。另外,根據表面疏水性胺基酸的分布,設計了L176E、L184E與Y252E的突變株,L184E的PHB降解活性只剩野生株的15%,推測Leu184是與PHB結合的重要胺基酸之一。另外在BtPhaZ晶體結構中觀察到七個ethylene glycol形成之片段,此片段會與Ser102形成氫鍵,以及與Met38、Trp101、Met103、Tyr133、Val166、Tyr177、Val181、Trp182、Leu184、Leu185、Ile186與Val253形成疏水性作用。未來可用單點突變法來找到與PHB結合的重要胺基酸,改造這些胺基酸,希望能提高酵素催化效率。
Trehalose synthase (TS)催化廉價的麥芽糖轉化成高價值的海藻糖,此異構化反應具有高專一性、高轉換率與不需輔酶和能量等優點,因此TS是一個具有開發價值的工業酵素。由本實驗室林易霆培養DrTS蛋白晶體,並收集2.7 Å解析度的X光繞射數據,我則使用分子取代法測定出DrTS與其抑制劑Tris的複合結構。DrTS屬於glycoside hydrolase family 13 (GH13),主要由一個典型GH13 catalytic (/)8 barrel、subdomain B與domain C組成,另外還含有TS特有的S7與S8 subdomains。與其相似結構比對分析,發現這些較封閉構型都具有相似催化中心,及相似subdoamain B的方位,由此推論DrTS-Tris是因抑制劑結合而形成的封閉構型,在DrTS-Tris結構中,subdomain B與S7形成許多交互作用,而關閉了催化中心,促使麥芽糖進行分子內異構化而形成海藻糖。將Arg148與Asn253突變成alanine以減少交互作用力的形成,而此兩突變株的異構化活性下降8-9倍,水解活性則上升1.5-1.8倍。N253A晶體結構顯示,蛋白表面具有小孔洞,讓水分子得以進入活性中心,而水解反應中間產物。此外DrTS-Tris與NpAS-maltoheptoase似乎具有幾乎相同的麥芽糖結合位置,因而可建立DrTS-maltose模擬結構,Tyr213、Glu320與Glu324可能與+1 site葡萄糖形成氫鍵,Y213A、E320A與E324A突變株都沒有偵測到活性。最後,DrTS-maltose結構也建議了一些可能參與於受質與反應專一性的胺基酸,而晶體結構也顯示四聚體的可能形成,因此改造這些相關胺基酸或許能製備更高效能的突變株。

An intracellular poly-3-hydroxybutyrate depolymerase from Bacillus thuringiensis (BtPhaZ) and a trehalose synthase from Deinococcus radiodurans (DrTS) have been screened for potential inductrial applications in polyester biodegradation and trehalose production, respectively. In this dissertation, I first determined the crystal structures of these two enzymes by the X-ray diffraction method and then performed structural-based mutational analysis in order to understand the ezyme mechanism and substrate specifity. Hopefully, my studies would provide useful information for development of more efficient enzymes for potential industrial applications.
BtPhaZ catalyzes the degradation of poly-3-hydroxybutyrate into 3-hydroxy- butyrate (3HB). The recombinant protein has been crystallized and the X-ray diffraction data have been collected at 1.42 Å resolution by Chen Chia-Lin. Here I solved the BtPhaZ structure using the molecular replacement method even BtPhaZ only shares 24% sequence identity to the proteins in the PDB database. BtPhaZ consists of a canonical /hydrolase fold and a helical domain. A detailed structural comparison reveals three new conserved signatures, HG36, D61xxGxG and G248xxD, in addition to the most conserved signature in the /hydrolase superfamily, GxS102xG. Three mutants including G36A, D61A and G248A were generated and characterized. The turbidimetric assay revealed that G36A and G248A displayed 5% and 23% activities of the wild type, respectively. The esterase activity assay showed that G36A displayed a 10,000-fold decrease in kcat compared to the wild type. The decreased activities of G36A and G248A may be due to unfavorable contacts with surrounding residues such as Trp101 and Cys277, respectively. The D61A mutant was expressed in the inclusion body, suggesting that the extensive interactions between Asp61and Ser40, Ser41, and Asn67 are essential for the structural integrity. Therefore, these four conserved signatures not only constitute the catalytic triad and the oxyanion hole, but also attain the active-site conformation. In addition, a 3HB trimer was modeled into the active site with subsequent mutational analysis. The turbidimetric assay revealed that T39V, Y133F, Y133A and N214A showed a similar hydrolytic activity to the wild type, while N37A and N37D retained 20% activity. Activity assays were consistent with the complex model, in which Asn37Nwas proposed to interact with the carbonyl group of the 3HB at the +2 stie. Moreover, a cluster of exposed hydrophobic residues may be responsible for the PHB attachment, including Val146, Leu149, Val161, Leu176, Leu184, Tyr252, Val253 and Val257. The L176E, L184E and Y252E mutants were first generated, and the turbidimetric assay revealed that L184E displayed 15% activity of the wild type, thus Leu184 is important for the PHB attachment. Furthermore, a putative fragment containing seven units of ethylene glycol was observed at the active site, which forms hydrophobic contacts with Met38, Trp101, Met103, Tyr133, Val166, Tyr177, Val181, Trp182, Leu184, Leu185, Ile186 and Val253. In the future, more mutants will be generated to approach the polymer binding, and hopefully, the mutants with higher PHB affinities will be obtained.
DrTS catalyzes the conversion of the inexpensive maltose to trehalose with high substrate specificity and high conversion rates. The recombinant protein has been crystallized by Lin Yi-Ting and the X-ray diffraction data have been collected at 2.7 Å resolution. I determined the dimeric DrTS-Tris structure by the molecular replacement method. DrTS belongs to glycoside hydrolase family 13, and consists of a catalytic (/)8 domain, subdomain B, domain C and two TS-unique subdomains (S7 and S8). The domain C and S8 contribute the majority of the dimeric interface. DrTS shares high structural homology with sucrose hydrolase, amylosucrase, and sucrose isomerase in complex with sucrose, in particular a virtually identical active-site architecture and a similar substrate-induced rotation of subdomain B. The interaction networks between subdomain B and S7 seal the active-site entrance that will facilitate intramolecular isomerization and minimize disaccharide hydrolysis. Disruption of such networks through the replacement of Arg148 and Asn253 with alanine resulted in a decrease in isomerase activity by 89-fold and an increased hydrolase activity by 1.51.8-fold. The N253A structure showed a small pore created for water entry. Interestingly, the nonreducing terminal maltosyl residue in the NpAS-maltoheptoase complex fits the -1 and +1 site in DrTS-Tris well, and hence a DrTS-maltose structure was modeled. This model suggested the Tyr213, Glu320 and Glu324 form hydrogen bonds to the glucose unit at the +1 site. Neither isomerase nor hydrolase activity was detected when the Y213A, E320A and E324A mutants wre examed. Finally, the DrTS-maltose structure suggested that some residues are involed in substrate and reation specificity. Also modification of several residues may result in formation of more stable tetramer. Hopefully, we can gain some mutants with high catalytic efficiency and high thermostability.

中文摘要...............................................I
英文摘要..........................................IV
目錄..................................................VII
表目錄.................................................X
圖目錄.....................................................XI

第一部分、Poly-3-hydroxybutyrate depolymerase from Bacillus thuringiensis..1
第一章、緒論..........................................2
一、Polyhydroxyalkanoate (PHA)與poly-3-hydroxybutyrate (PHB) .........2
二、PHB之生合成與降解......................................2
三、PHB降解酶 (PHB depolymerase) ...........................3
四、/ hydrolase superfamily.............................4
五、 PhaZs已知晶體結構...................................5
六、BtPhaZ.........................................6
七、研究目的...........................................7
第二章、材料與方法.....................................8
一、突變株之選殖與重組蛋白之備製......................8
二、PHB granules之備製...................................9
三、活性分析方法.....................................9
四、分析型超高速離心實驗..................................10
五、晶體結構之測定過程...................................10
第三章、結果與討論.......................................11
一、BtPhaZ晶體結構測定...................................11
二、BtPhaZ之蛋白結構......................................11
三、BtPhaZ與/ hydrolase superfamily成員之結構比對........12
四、高度保留區之功能研究............................13
五、BtPhaZ-3HB trimer之模擬複合結構..................14
六、BtPhaZ-heptaethylene glycol複合結構: PHB結合位置......16
七、BtPhaZ雙聚體之形成可能性..................17
八、結論..................................18
第四章、參考文獻..................................19
第五章、圖表..................................24

第二部分、Trehalose synthase from Deinococcurs radiodurans.....49
第一章、緒論.............................................50
一、海藻糖之生物特性與功能.............................50
二、海藻糖之應用.........................................50
三、海藻糖之生物合成.....................................51
四、海藻糖合成酶 (trehalose synthase) ......................51
五、研究目的...........................................52
第二章、材料與方法......................................54
一、重組蛋白之備製........................................54
二、DrTS晶體培養與繞射實驗................................54
三、分析型超高速離心實驗..................................55
四、活性分析方法..........................................55
五、晶體結構之測定過程.....................................55
第三章、結果與討論........................................57
一、DrTS晶體結構測定.....................................57
二、DrTS晶體結構.......................................57
三、DrTS與其它GH13成員之結構比較........................58
四、TS之不同聚合狀態...................................59
五、DrTS之金屬結合位置................................60
六、相似之催化中心.....................................61
七、受質結合中心......................................62
八、受質結合造成構型改變....................................63
九、TS之開放與封閉構型................................64
十、DrTS蛋白工程之突變株設計...........................65
十一、結論.........................................65
第四章、參考文獻...........................................67
第五章、圖表................................................72


表目錄
第一部分、Poly-3-hydroxybutyrate depolymerase from Bacillus thuringiensis
表一、BtPhaZ晶體繞射數據與結構細調參數.................24
表二、突變株引子設計...............................25
表三、分子取代法之模板與最佳解...........................26
表四、BtPhaZ結構相似之/ hydrolase superfamily成員........27
表五、 野生株、G36A與G248A突變株之活性分析..............28
表六、野生株與N37D突變株在pH 7.5和pH 9下之酵素動力學常數分析...29

第二部分、Trehalose synthase from Deinococcurs radiodurans
表一、DrTS野生株與N253A突變株之晶體繞射數據與結構細調參數.......72














圖目錄
第一部分、Poly-3-hydroxybutyrate depolymerase from Bacillus thuringiensis
圖一、PHB granuls..................................................30
圖二、PHB的生合成與降解..........................................31
圖三、PlPhaZ7-3HB trimer與PfPhaZ-3HB trimer之二級與三級結構......32
圖四、BtPhaZ與EG7複合晶體結構...................................33
圖五、不對稱單元中四個BtPhaZs結構重疊............................34
圖六、BtPhaZ與 hydrolase superfamily成員類似之catalytic domains結構
比對..............................................................35
圖七、BtPhaZ與三個 hydrolase superfamily成員之類似helical domains結構比對............................................................36
圖八、高度保留區之胺基酸交互作用..................................37
圖九、BtPhaZ與三個hydrolase fold superfamily成員之活性區域結構重疊................................................................38
圖十、BtPhaZ與3HB trimer之模擬複合結構..........................39
圖十一、N37A、N37D突變株與野生株在不同pH值下之PHB降解活性分析................................................................40
圖十二、PlPhaZ7、PfPhaZ與BtPhaZ之表面電位圖和iserted segments之結構
比對..............................................................41
圖十三、BtPhaZ之PHB結合區......................................43
圖十四、BtPhaZ與EG7之交互作用...................................44
圖十五、PEG 400對 BtPhaZ抑制能力................................45
圖十六、BtPhaZ晶體結構中的雙聚體.................................46
圖十七、BtPhaZ分析型超高速離心實驗...............................47
圖十八、BtPhaZ單分子與雙聚體之表面疏水性胺基酸之分布.............48
第二部分、Trehalose synthase from Deinococcurs radiodurans
圖一、海藻糖五種生合成路徑.........................................73
圖二、MsTS可能之異構化機制........................................74
圖三、DrTS野生株與N253A突變株晶體結構比較.......................75
圖四、DrTS之雙聚體與單分子晶體結構................................76
圖五、DrTS與GH13成員之結構重疊..................................77
圖六、以結構為基礎之序列比對.......................................78
圖七、DrTS分析型超高速離心實驗....................................79
圖八、雙聚體間之交互作用...........................................80
圖九、相似之葡萄糖結合-1 site........................................81
圖十、催化三元體鄰近胺基酸之交互作用網路...........................82
圖十一、Tris對DrTS抑制能力.......................................84
圖十二、受質結合中心.............................................. 85
圖十三、受質結合引起結構改變...................................... 86
圖十四、TSs與SHs類似之受質結合引起結構改變...................... 88
圖十五、TSs的開放構型與封閉構型................................... 90
圖十六、DrTS可能之異構化機制..................................... 92
圖十七、DrTS、RhSI與ScIM之糖結合+1 site胺基酸結構比對........... 93

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