請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/31197
標題: | 酵母菌四異戊二烯焦磷酸合成酶及大腸桿菌十一異戊二烯焦磷酸合成酶之三度空間結構及反應機制之研究 Structural Study of Geranylgeranyl Pyrophosphate Synthase from S. cerevisiae and Undecaprenyl Pyrophosphate Synthase from E. coli |
作者: | Rey-Ting Guo 郭瑞庭 |
指導教授: | 王惠鈞(Andrew H.-J. Wang) |
關鍵字: | 類異戊二烯,蛋白質結構,同步輻射,抗生素, isoprenoid,protein structure,synchrotron radition,antibiotic, |
出版年 : | 2007 |
學位: | 博士 |
摘要: | 類異戊二烯族 (isoprenoid) 為廣泛分布於自然界中的化合物,是由異戊二烯轉移酵素 (prenyltransferase) 催化異戊二烯焦磷酸 (isopentenyl pyrophosphate) 為合成骨架所構成的聚合物。其中一類的酵素催化含多個異戊二烯焦磷酸 (5個碳) 和法呢基焦磷酸 (15個碳) 結合生成長鏈產物,並依生成產物之立體結構命名為反式 (trans-) 或順式 (cis-) 類異戊二烯轉移酵素。本論文中所研究的反式酵素為酵母菌第三型-四異戊二烯焦磷酸合成酶 (geranylgeranyl pyrophosphate synthase),產物是四異戊二烯化 (geranylgeranylated) 蛋白質、胡蘿蔔素 (carotenoid)、細胞膜脂質等等…這些在生物體內重要分子的前趨物。
而在本論文中所研究的順式酵素為大腸桿菌的十一異戊二烯焦磷酸合成酶 (undecaprenyl pyrophosphate synthase),產生的 55 個碳的十一異戊二烯焦磷酸 (undecaprenyl pyrophosphate) 可攜帶醣質以合成細菌的細胞壁 (cell wall),對細菌的存活非常的重要,也因此許多藥廠針對發展此酵素之抑制劑來做為新型的抗生素藥物的研發目標。 藉由不同的類異戊二烯轉移酶,可以合成出不同長度的直鏈產物,並在生物體內皆扮演不同的生理角色,因此這一類的酵素必須非常精準地調控其直鏈產物的長短,而比較這些合成不同長度的酵素的三度空間結構將有助於我們了解這一類酵素調控產物長短的機制。因此,我們之前也解出並發表了合成 40 個碳的反式長鏈產物的八異戊二烯焦磷酸合成酶 (octaprenyl pyrophosphate synthase) 的 X 光晶體結構,分析此酵素的三度空間結構後,我們發現在活性區下有一空洞可供產物生成,我們並確認了在空洞下方的苯丙胺酸 (Phe132) 為決定鏈長最重要的胺基酸,若再將空洞往下會遇到的大的胺基酸 (Leu128, Ile123 及 Asp62) 改成小的胺基酸 (Ala),則產物甚至可達到 95 個碳的長度。 而比較反式和順式的酵素的胺基酸序列,可以了解到這兩種酵素是分屬完全不同的兩種酵素,雖然在催化反應進行時,反式及順式酵素需要同樣的受質 (異戊二烯焦磷酸 (IPP) 和法呢基焦磷酸 (FPP) 以及二價鎂離子),但是這兩種酵素所使用的催化活性區卻是完全不同的。為了完全了解這兩種酵素在催化機制上的差異,因此我們將催化反應需要的受質 (異戊二烯焦磷酸 (IPP) 和法呢基焦磷酸 (FPP) 以及二價鎂離子) 以浸泡或共結晶的方式,因而得到酵母菌第三型-四異戊二烯焦磷酸合成酶及大腸桿菌十一異戊二烯焦磷酸合成酶和這些受質的複合結構,並得以了解反式及順式酵素之間的異同。 在抑制劑的研究方面,我們和美國伊利諾大學 Eric Oldfield 教授合作,Oldfield 教授是致力發展新型雙磷酸鹽類 (Bisphosphonates) 的專家學者,雙磷酸鹽類的結構和焦磷酸鹽類 (Pyrophosphates) 極為相似,其結構中之PCP基團為其生物活性骨架,是與氫氧磷灰石 (Hydroxyapatite) 鍵結所必須之結構基團,對骨骼具有強烈親和力。目前臨床上除了骨質疏鬆症外,惡性腫瘤骨轉移 (以前列腺癌、乳癌和肺炎最常見) 及高血鈣也常使用雙磷酸鹽類。在本篇論文中,我們解出 5 個新型雙磷酸鹽類和酵母菌第三型-四異戊二烯焦磷酸合成酶的複合結構,及 5 個新型雙磷酸鹽類和十一異戊二烯焦磷酸合成酶的複合結構,而分析這些雙磷酸鹽類和反式、順式酵素的三度空間結構,可發現許多有趣的鍵結模式,這些結構上的資訊也將提供我們許多設計新型藥物或抗生素的寶貴經驗。 Over 23000 compounds constitute an important family of natural products named isoprenoids, which are built on the 5-carbon isopentenyl pyrophosphate (IPP). Isoprenyl pyrophosphate synthases (IPPs) involved in the biosynthesis of the linear isoprenoid polymers each catalyzes consecutive condensation reactions of a designated number of isopentenyl pyrophosphate (IPP) with a single farnesyl pyrophosphate (FPP). These enzymes are named trans- or cis-prenyltransferases according to the stereochemistry of the double bonds from IPP condensation. Among trans-prenyltransferases, geranylgeranyl pyrophosphate synthase (GGPPs) catalyzes a condensation reaction of farnesyl pyrophosphate (FPP) with an isopentenyl pyrophosphate (IPP) to generate C20 geranylgeranyl pyrophosphate (GGPP), a precursor for carotenoids, chlorophylls, geranylgeranylated proteins, and archaeal ether linked lipid. In this study, we solved 6 crystal structures of S. cerevisiae GGPPs in complex with substrate FPP, with IPP and the substrate analog farnesyl thiopyrophosphate (FsPP), alternative substrate GPP, and product GGPP. Unlike in FPP synthase where the third Mg2+ ion coordinated by the second DDxxD motif participates in the substrate binding, only two Mg2+ ions are observed in S. cerevisiae GGPPs. IPP is bound in a positively-charged pocket with Arg39, His68, Arg85 and Tyr205. From the binding mode of the product GGPP, the pyrophosphate of GGPP binds to the IPP pyrophosphate binding site and the hydrocarbon moiety sits in the FPP hydrocarbon binding site. The binding mode for GGPP in yeast GGPPs structure is distinct from that of the product in the proposed inhibitory site of human GGPPs. Bisphosphonate drugs used for osteoclast-mediated bone resorption and tumor-induced hypercalcemia are potent inhibitors of farnesyl pyrophosphate synthase (FPPs). Both FPP and GGPP are essential ligands for posttranslational modification of small GTPases such as Ras, Rac, and Rho for their biological functions. To design specific inhibitors for GGPPs, we have solved five S. cerevisiae GGPPs-bisphosphonates complex structures in this thesis (These bisphosphonates are kindly provided by Prof. Eric Oldfield). Unlike the previous finding that bisphosphonate inhibitors bind to the essential Mg2+ ions in the FPPs active site, these inhibitors also bind to GGPPs, but not necessary with Mg2+ ion. The bisphosphonates can bind to GGPPs in the FPP site, IPP site and/or the product feedback inhibitory site. Moreover, particular bisphosphonates with hydrophobic moiety bind to undecaprenyl pyrophosphate synthase, which is a cis-prenyltrasnferase and catalyzes chain elongation of FPP by condensation with 8 IPP to form C55 product as lipid carrier for bacterial peptidoglycan biosynthesis. Cis-prenytransferases share no sequence homology and structural similarity with the trans-enzymes. In each of the five structures investigated, we found that there were up to four binding sites per monomer. Three of the binding sites occupy the top of a “funnel” region, while the fourth site is situated at the bottom of the funnel. One inhibitor molecule occupies the FPP substrate binding site and none competes with IPP binding. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/31197 |
全文授權: | 有償授權 |
顯示於系所單位: | 生化科學研究所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-96-1.pdf 目前未授權公開取用 | 11.68 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。