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  • 學位論文

金屬膜蛋白對碳氫化合物之代謝作用

Metabolism of Hydrocarbon Compounds By Membrane-Bound Metalloenzymes

指導教授 : 張繼堯
共同指導教授 : 俞聖法(Steve Sheng-Fa Yu)
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摘要


金屬酵素為生物體細胞中重要的催化反應酵素,而其中又以帶有非血基質雙鐵中心的蛋白最為重要。第三類型非血基質雙鐵中心金屬酵素包括膜結合型脂肪酸去飽和酶超級家族之蛋白,對環狀、飽和烷烴類及不飽和烷烴類化合物進行去飽和及單加氧催化反應。 斑馬魚Δ-5/Δ-6脂肪酸去飽和酶(Z-FADS)在長鏈不飽和脂肪酸( long-chain polyunsaturated fatty acids, LC-PUFAs)催化與生合成過程中扮演重要角色。以螢光共振能量轉移(fluorescence resonance energy transfer, FRET)證明斑馬魚Z-FADS蛋白會與第二及第三型細胞色素b5還原酶(cytochrome b5 reductase, CYB5R2及CYB5R3)及四種延長酶(elongation of very long chain fatty acids, ELOVL, ELOVL2, 4, 5及7)產生交互作用。將斑馬魚fads、elovl2、elovl4及elovl5基因同時轉染至HeLa細胞,以α-次亞麻油酸(α-linolenic acid, ALA)為反應起始物,以氣相層析圖譜分析法,證明斑馬魚ω3系列長鏈不飽和脂肪酸生合成過程中,特別是DHA及EPA的生合成需要Z-FADS、ELOVL5、ELOVL2及ELOVL4的協同作用。此外以西方墨點法及免疫螢光染色分析,也證明斑馬魚Z-FADS除了在內質網表現外,亦會存在於細胞的粒線體中。為了瞭解金屬酵素之催化反應機制,以同屬於膜結合型脂肪酸去飽和酶超級家族之原核生物蛋白─二甲苯單加氧酶為模式進行研究。免疫共沉澱法及免疫金標誌分析證明,在催化反應過程中,還原酶會與催化酵素本體結合而形成複合體,而未形成複合體之電子傳遞本體會出現在細胞質中靠近細胞膜處,且證明在催化過程中,催化酵素本體與電子傳遞本體比例不等量,催化酵素本體多於電子傳遞本體。以二甲苯單加氧酶對反應物催化活性分析,證明二甲苯單加氧酶除了會催化苯環類化合物,亦會對短鏈不飽和烷烴類化合物進行催化反應,而含氟化合物更證明,含氟化合物更可改善催化反應效率。 本論文同時以真核及原核生物之膜結合型脂肪酸去飽和酶為研究主軸,分析金屬酵素在催化反應過程中,反應口袋與反應物之間的交互作用。證明斑馬魚Z-FADS在DHA及EPA生合成過程中所扮演之角色,同時透過二甲苯單加氧酶為模式更證明,疏水性反應口袋可有效的對不同反應物進行特定部位的催化反應。

並列摘要


The membrane metallo-proteins (MMPs) are important catalytic enzyme in organism, particularly the non-heme diiron containing proteins. Membrane-bound fatty acids desaturase superfamily proteins (membrane-FADS superfamily proteins) are classified into type Ⅲ non-heme diiron center proteins that catalyze desaturation and/or monooxygenation in hydrocarbons including aromatics, saturated and unsaturated. Zebrafish Δ-5/Δ-6 fatty acid desaturase (Z-FADS) catalyzes cascade synthesis of long-chain polyunsaturated fatty acids (LC-PUFAs) and plays pivotal roles in many biological functions. In this study, we deployed the technique of fluorescence resonance energy transfer (FRET) to examine the protein-protein interactions between Z-FADS and cytochrome b5 reductases (CYB5R1-3), elongases 2, 4, 5, 7 (elongation of very long chain fatty acids, ELOVL), respectively and the results indicated that, in endoplasmic reticulum (ER), the Z-FADS can be in close proximity to CYB5R2, 3, and ELOVL protein family, including ELOVL2, 4, 5 and 7, respectively. Furthermore, in the gas chromatography analysis, we proved that the HeLa cells co-transfected with fads, elovl2, 4 and 5 catalyze α-linolenic acid to ω3-series LC-PUFAs, especially the docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Additional data from immuno-fluorescence cytochemistry (IFC) and Western blotting studies demonstrated that Z-FADS also resides in the mitochondria of zebrafish fads transfected HeLa cells. Our results implicated that Z-FADS, the sole fatty acid desaturase ever been identified in zebrafish, can serve as a universal fatty acid desaturase for the whole lipogenesis process. Xylene monooxygenase (XylM), a non-heme diiron monooxygenase from the prokaryotic system, Pseudomonas putida mt-2, can catalyze toluene to benzyl alcohol and xylene to 4-methylbenzyl alcohol. An E. coli system heterogeneously transformed with part of an TOL operon including the overexpression of XylM and its partner protein of oxidoreductase, XylA, is constructed for its application of whole-cell catalysis. The protein expressions of XylM and XylA in this system are verified by the studies of Western blotting analysis, co-immune-precipitation as well as transmission electron microscopy (TEM) with immune-gold staining experiments. Several substrates including fluorinated butane are designed to study their enzymatic activities. Our results indicated that the aromatic residue(s) within this metalloprotein play important roles for its controlled hydroxylation. In overall, I exploited both the eukaryotic and prokaryotic membrane-bound non-heme iron proteins including FADS superfamily as my target proteins and pave my way towards understanding the role of Z-FADS on DHA and EPA biosynthesis in Zebrafish as well as showing the promiscuity of hydrophobic pocket in xylene monooxygenase can efficiently achieve selective oxidations in a variety of substrates.

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