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研究生:張珮珊
研究生(外文):Pei-Shan Chang
論文名稱:豌豆蚜內共生菌Buchnerasp.胺基酸合成酵素AroQ-PheA之動力學特性
論文名稱(外文):Kinetic Properties of the Amino-acid Biosynthesis Enzyme AroQ-PheA from Buchnera sp. APS
指導教授:賴吉永賴吉永引用關係
指導教授(外文):Chi-Yung Lai
學位類別:碩士
校院名稱:國立彰化師範大學
系所名稱:生物學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:95
中文關鍵詞:蚜蟲共生菌芳香族胺基酸苯丙胺酸回饋抑制
外文關鍵詞:AphidBuchnerasymbiosisP-proteinAroQPheAchorismate mutaseprephenate dehydratasephenylalaninefeedback inhibition
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蚜蟲分類為半翅目( Hemiptera ),蚜蟲總科 ( Aphidoidea ),以刺吸式口器吸食植物汁液維生,對農業影響甚鉅,然韌皮部含氮養份不足,故蚜蟲體內具共生菌Buchnera 饋補此營養需求。Buchnera分類為γ- Proteobacteria中的腸內細菌科 ( Enterobacteriacae ),與大腸桿菌 ( Escherichia coli ) 親源關係相近,主要供給蚜蟲必需胺基酸養份。三種芳香胺基酸中,蚜蟲所需之苯丙胺酸 ( phenylalanine )、色胺酸 ( tryptophan ) 衍生自Buchnera,又以phenylalanine 含量較高,也正因此胺基酸合成與供應的共生關係,推測Buchnera胺基酸代謝調節應不似其他生物嚴謹。Buchnera基因體中,含合成phenylalanine 關鍵酵素基因 pheA,編碼具有chorismate mutase ( CM;AroQ )、 prephenate dehydratase ( PDT;PheA ) 雙催化功能之異位調節酵素P- protein。 pheA上游缺乏減弱子,故無基因層級之調節,終產物 phenylalanine回饋抑制的酵素調控層面,Escherichia coli P- protein調節功能區 ( ACT domain ) 保守之關鍵殘基ESRP是回饋調控重要位點,但Buchnera P- protein此區序列為TSQK,顯示其phenylalanine 代謝可能亦不具酵素層級調控。本實驗利用功能互補試驗,證實 Buchnera pheA 在 Escherichia coli 表現系統中,具有 AroQ、 PheA 之催化活性,並透過低溫表現條件、純化與活性試驗,進行 Buchnera P- protein 之動力學分析,Buchnera AroQ 之 Km 為 459.9±87.9 μM,PheA 之 Km 為 594.8±132.7 μM ( stop assay ) 或1382.6±103 μM ( online assay ) ;終產物回饋抑制層面,Buchnera AroQ 活性不受 phenylalanine 影響,而PheA 活性受 phenylalanine之最大抑制效率僅為 8.3%,I0.5 約 17.79 μM, Ki 為 17.77 μM ,證實 Buchnera P- protein 對 phenylalanine 的回饋抑制甚不敏感,酵素層級調節效率低落,而具大量合成 phenylalanine 之特性; Buchnera AroQ 、 PheA 活性亦不受 tyrosine 影響, tryptophan 對 Buchnera PheA 亦無明顯抑制,但卻能活化Buchnera AroQ,使得其能過量合成 phenylalanine,一方面提供宿主蚜蟲利用,部分則透過蚜蟲懷菌細胞進一步代謝為 tyrosine,滿足蚜蟲與 Buchnera 本身之需求,以維持此共生關係。
Aphids belong to the superfamily Aphidoidea of the order Hemiptera. Aphids feed on the nutritionally poor diet of phloem sap, and thus are agricultural pests. These insects maintain an endosymbiotic association with Buchnera, a γ- Proteobacterium closely related to Escherichia coli. The main role of the symbionts is the provision of essentail amino acids that the cannot be synthesized by the host and are scarce in the phloem- sap diet. Among the three aromatic amino acids, most of aphid’s need for phenylalanine and tryptophan are met by Buchnera, especially the need for phenylalanine. To fulfill their nutritional role, Buchnera cannot place a tight regulation on phenylalanine biosynthesis. Regulation of phenylalanine biosynthesis can be achieved through the control of gene expression and enzyme activity. The pheA encoded P- protein (AroQ-PheA) is a bifunctional, allosteric enzyme playing critical regulatory role in enteric bacteria. The upstream region of Buchnera pheA does not have any sequence resembling an attenuator indicating a lack of transcriptional control. At the enzyme level, the dual activies of chorismate mutase (CM, AroQ) and prephenate dehydratase (PDT, PheA) are negatively regulated in Escherichia coli by phenylalanine, which binds to the ACT domain of P- protein. However, an important feature of the ACT domain of the Buchnera protein is the lack of a conserved four- residue sequence (ESRP), which is changed to TSQK. Due to these changes, Buchnera P- protein (AroQ-PheA) were proposed to be insensitive to feedback inhibition by phenylalanine. I examed the Buchnera pheA gene function by testing the enzyme activities of AroQ and PheA in Escherichia coli using functional complementation tests, I them expressed pheA under low-temperature conditions and partially purified the enzyme. Analysis of the Michaelis-Menten kinetics of the enzyme showed that the Km of AroQ was 459.9±87.9 μM , and the Km of PheA was 594.8±132.7 μM (stop assay) or 1382.6±103 μM (online assay). As for the properties of end- product inhibition, phenylalanine had no obvious effect on Buchnera AroQ activity, and inhibited PheA activity by only 8.3%, with an I0.5 of about 17.79 μM, and a Ki of 17.77 μM. These values indicated an insensitivity of Buchnera P- protein to phenylalanine and a lack of regulation at the enzyme level, properties in favor of phenylalanine overproduction. Furthermore, I found no effect of tyrosine either to Buchnera AroQ or PheA activities. Tryptophan had no effect on PheA, but it activated AroQ. These properties enable Buchnera to overproduce and supply phenylalanine to aphids. Some phenylalanine is converted to tyrosine by bacteriocytes of aphids and retured to Buchnera. These results showed the interdependence between aphids and Buchnera in keeping this symbiotic relationship.
目錄
中文摘要 I
英文摘要 II
誌謝 Ⅳ
前言 1
文獻探討
一、蚜蟲 4
二、蚜蟲與內共生菌. 6
三、內共生細菌Buchnera基因組特性 10
四、蚜蟲與內共生菌Buchnera之間的營養關係 11
五、芳香族胺基酸合成途徑與合成酵素 13
六、苯丙胺酸合成之調控機轉 17
材料與方法 22
結果
一、Buchnera pheA 基因於突變株 KA12 功能互補測試 31
二、Buchnera P- protein ( AroQ-PheA ) 於 CAG12158 中表現條
件與酵素活性 ( PheA ) 測試 31
三、Buchnera P- protein ( AroQ-PheA ) 於 KA12 中不同溫度之表
現、純化與酵素活性 ( AroQ、PheA ) 測定 32
四、AroQ-PheA Steady-State Kinetic 33
五、AroQ-PheA 終產物回饋抑制分析. 34
六、Buchnera PheA ( BuPDT ) 結構模擬35
討論 37
結論 44
參考文獻 45

表目錄
表一、Chorismate mutase ( CM ) 之 Km 與測試條件.55
表二、Prephenate dehydratase ( PDT) 之 Km 與測試條件. 56

圖目錄
圖一、Buchnera pheA 基因於突變株 KA12 功能互補測試. 57
圖二、Buchnera P- protein ( AroQ-PheA ) 於CAG12158 之表現與
酵素活性測定 58
圖三、Buchnera P- protein (AroQ-PheA) 於KA12不同溫度( 37℃、
20℃ ) 之表現、純化. 60
圖四、Buchnera AroQ 於 KA12 不同溫度 ( 37℃、20℃ ) 表現之活
性測定 61
圖五、Buchnera PheA 於 KA12 不同溫度( 37℃、20℃ ) 表現之活性
測定 62
圖六、Escherichia coli AroQ 、 Buchnera AroQ Steady-State
Kinetic. 63
圖七、Escherichia coli PheA 、 Buchnera PheA Steady-State
Kinetic. 64
圖八、Buchnera PheA Steady-State Kinetic 65
圖九、Phenylalanine 對 Escherichia coli AroQ、Buchnera AroQ
之回饋抑制 66
圖十、Phenylalanine 對 Escherichia coli PheA、Buchnera PheA
之回饋抑制 67
圖十一、Phenylalanine、trytophan、tyrosine 對 Buchnera AroQ 活
性之影響 68
圖十二、Phenylalanine、trytophan、tyrosine 對 Buchnera PheA
活性之影響 69
圖十三、Phenylalanine 對 Buchnera PheA 抑制之 Ki70
圖十四、Buchnera ( BuPDT ) 與 Chlorobium tepidum
( CtPDT )序列比對71
圖十五、CtPDT 與 BuPDT 立體構型 ( Superimpose )比對. 72
圖十六、Buchnera ( BuPDT ) 與 Staphylococcus aureus
( SaPDT ) 序列比對73
圖十七、SaPDT 與 BuPDT 立體構型 ( Superimpose )比對 74
圖十八、PDTa Domain 與ACT Domain 間帶電性殘基之距離 75
圖十九、Buchnera sp. APS 芳香族胺基酸合成途徑與調控 76
附錄
附錄一、Phenylalanine 對 Buchnera PheA 活性抑制之 Ki 值計算
法 77

附錄圖
圖一、宿主植物-蚜蟲-共生菌 Buchnera 胺基酸養分供需關係 78
圖二、Escherichia coli之芳香族胺基酸合成途徑與調控 79
圖三、Prephenate dehydratase之立體結構
( Staphylococcus aureus, SaPDT ) 80
圖四、Prephenate dehydratase之立體結構 ( Chlorobium tepidum, CtPDT ) 81
圖五、異位調節區 ( ACT Domain) 之保守性殘基 ( CtPDT ) 82
圖六、SaPDT 與 CtPDT 活化位關鍵殘基Threonine 83
圖七、不同物種之 Prephenate dehydratase ( PDT ) 胺基酸序列比對 84
圖八、Ramachandran plot of BuPDT (2qmx) 85
圖九、Ramachandran plot of BuPDT (2qmw) 86
圖十、Saccharomyces cerevisiae之芳香族胺基酸合成途徑與調控 87
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