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dc.contributor.advisor梁博煌(Po-Huang Liang)
dc.contributor.authorTao-Hsin Changen
dc.contributor.author張道欣zh_TW
dc.date.accessioned2021-06-13T04:32:12Z-
dc.date.available2008-07-27
dc.date.copyright2006-07-27
dc.date.issued2006
dc.date.submitted2006-07-20
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dc.identifier.urihttp://tdr.lib.ntu.edu.tw/jspui/handle/123456789/33272-
dc.description.abstract類異戊二烯族 (isoprenoid) 為廣泛分布於自然界中的化合物,由一類異戊二烯轉移酵素 (trans-prenyl transferase) 所催化合成,並且亦是以異戊二烯焦磷酸 (isopentenyl pyrophosphate) 為骨架所構成的聚合物。本研究之酵母菌第三型-四異戊二烯焦磷酸合成酵素 (geranylgeranyl pyrophosphate synthase) 可催化一個異戊二烯焦磷酸與法呢基焦磷酸 (farnesyl pyrophosphate) 反應產生四異戊二烯焦磷酸 (geranylgeranyl pyrophosphate),此產物是四異戊二烯化 (geranylgeranylated)蛋白質、胡蘿蔔素 (carotenoid)、細胞膜脂質等-這些在生物體內重要分子的前趨物。不過藉由不同的一類異戊二烯轉移酵素,所合成出長度具多樣性的直鏈產物,分別在生物體內亦扮演不同的生理角色,因此此群酵素必須非常精準地調控其直鏈產物的長短。但是酵母菌第三型-四異戊二烯焦磷酸合成酵素不論從胺基酸系列比對,還是以已知結構的法呢基焦磷酸合成酵素為藍圖,皆無法推論出其可能調控直鏈產物的機制,而此即為本論文探討重點之一;此外,近年已有文獻報導以此纇酵素作為治療癌症的重要標的物之一。在本論文中,首度解出此酵素立體結構,為兩個相同單元體 (monomer) 所組成的二聚體 (dimer),每一個單元體由15個α螺旋 (helix) 並以環狀物 (loop) 所連接構成,其催化中心由兩個DDXXD motif所構成;同時意外發現鎂離子和活性中心兩個重要的胺基酸形成配位鍵,之後藉由動力學與螢光光譜的分析,證實鎂離子在催化反應中的聚合反應(condensation reaction) 扮演重要角色,而似乎不影響受質和酵素的親合。然於過去的研究中,指出合成約10到25碳數的短碳鏈一類異戊二烯轉移酵素,利用位於第一個DDXXD motif前面第四或第五個的芳香族胺基酸來調控其直鏈產物的長短;不過自胺基酸序列的比對中,卻發現第三型-四異戊二烯焦磷酸合成酵素沒有此特徵。因此利用酵素立體結構與定點突變,對調控產物鏈長之反應機制做進一步的探討,由研究結果證實第三型-四異戊二烯焦磷酸合成酵素的產物鏈長由Tyr107和His108這兩個重要殘基來調控。在立體結構的比較中,發現酵母菌第三型-四異戊二烯焦磷酸合成酵素其N端第一個α螺旋和之後的環狀物,其構形與方向和其他一類異戊二烯轉移酵素相異;而兩個單體間界面 (interface) 的高度保留殘基,為不具有極性的胺基酸Met111和也和多數一類異戊二烯轉移酵素利用芳香族胺基酸的π-π交互作用促進二元體的形成機制不同。本論文證實酵素N端的結構和兩個單體間界面共同決定二元體的形成;但為何具有與二元體相同活性區域的單元體,會失去酵素活性? 因此利用定點突變製造與無活性單元體△(1–17),擁有相同螢光的特質但保留活性的二聚體W15F做進一步的探討,之後再由螢光光譜分析和阻流反應分析儀 (stopped-floe) 來分析兩者之間的差異,自結果可初步推論單元體似乎仍具有與受質的結合能力但無法進行催化反應中的聚合反應;亦自實驗中發現酵母菌第三型-四異戊二烯焦磷酸合成酵素和兩個不同受質-異戊二烯焦磷酸和法呢基焦磷酸-的結合沒有先後順序。最後希望藉由本研究,對酵母菌第三型-四異戊二烯焦磷酸合成酵素在立體結構、反應機制和動力學有進一步的了解。zh_TW
dc.description.abstractGeranylgeranyl pyrophosphate synthase (GGPPs) catalyzes the condensation reaction of farnesyl pyrophosphate (FPP) with isopentenyl pyrophosphate (IPP) to generate C20 geranylgeranyl pyrophosphate, which is a precursor for carotenoids, chlorophylls, geranylgeranylated proteins, and archaeal ether linked lipid. For short-chain trans-prenyltransferases synthesizing C10–C25 products, bulky residues generally occupy the 4th or 5th position upstream from the first DDXXD motif to block further elongation of the final products. However, type-III GGPPs in eukaryotes lack any large residue at these positions. In this study, the first structure of homodimeric type-III GGPPs from Saccharomyces cerevisiae has been determined to 1.98-Å resolution. Each subunit is composed of 15 alpha-helices joined by connecting loops and is arranged with alpha-helices around a large central cavity. An elongated hydrophobic crevice surrounded by D, F, G, H, and I alpha-helices contains two DDXXD motifs at the top for substrate binding with one Mg2+ coordinated by Asp75, Asp79, and four water molecules. It is sealed at the bottom with three large residues of Tyr107, Phe108, and His139. Compared to the major product C30 synthesized by mutant H139A, the products generated by mutant Y107A and F108A are predominantly C40 and C30, respectively, suggesting the most important role of Tyr107 in determining the product chain length.
Distinct from other known structures of trans-prenyltransferases, the N-terminal 17 amino acids (9-amino acid helix A and the following loop) of the yeast GGPPs protrude from the helix core into the other subunit and contribute to the tight dimer formation. Deletion of the first 9 or 17 amino acids caused the dissociation of dimer into monomer and these two mutants of △(1–9) and △(1–17) showed a 300-fold decrease and abolished in enzyme activity, respectively. Unlike other trans-prenyltransferases usingπ-πstacking interactions to form dimer, we also identified Met111 residue on the highly conserved helix F in the interface between two subunits of GGPPs to be the essential for dimer formation. Consequently, the replacement Met111 with Glu resulted in a shift form dimer to monomer for the M111E mutant enzyme and about 3.5-fold decrease in catalytic activity. The replacement of Met111 with Phe to create a hydrophobic dimer interface brought the monomeric △(1–9) mutant to a partial tetramer and 15-fold increase in catalytic activity compared with △(1–9). These results suggest the N-terminal helix and the critical residue in the interface region may both contribute to the dimer formation.
To investigate the differences in the catalytic properties between dimer and monomer, the site-directed mutagenesis, fluorescence assay, and stopped-flow experiments were performed. One of two fluorescent Trp resides in GGPPs primary sequence, Trp15, was changed to Phe to create W15F to monitor the fluorescence change of Trp148 during the substrate binding and reaction. The monomeric △(1–17) also containing Trp148 served for comparison. Similar protein intrinsic fluorescence change was observed upon addition of FPP and IPP for the W15F and △(1–17). According to this preliminary study on fluorescence spectrophotometer assay and stopped-flow experiments, the monomer without catalytic activity may lose the performance on the condensation reaction, one step of the catalytic reaction. Based on the fluorescence measurements, GGPPs bind with FPP and IPP in a random binding order. The Mg2+ concentration dependence of the catalytic rate by GGPPs shows that the activity is maximal at [Mg2+] = 5 mM, but drops significantly when [Mg2+] = 50 mM.
In summary, our results provide a thorough understanding of S. cerevisiae type-III GGPPs in its structure, mechanism and kinetics, in terms of product chain length determination, dimer formation, substrate induced protein conformational change, and the role of Mg2+ ion in catalysis.
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Previous issue date: 2006
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dc.description.tableofcontents中文摘要 1
ABSTRACT 3
INTRODUCTION 6
1-1 Characteristics and Biosynthesis of Isoprenoid 7
1-2 Classification of Prenyltransferases 7
1-3 Isoprenyl Pyrophosphate Synthases 8
1-4 Mechanism of Product Determination of trans-prenyltransferases 9
1-5 Mechanism of Dimer Formation 10
1-6 Specific Aims of This Study 11
MATERIAL AND METHODS 15
2-1 Chemicals 16
2-2 Expression and Purification of GGPPs 16
2-3 Preparation of Selenomethionine-labeled GGPPs 17
2-4 Mass Spectrometry Analysis 18
2-5 Crystallization and Data Collection 18
2-6 Structure Determination and Refinement 19
2-7 Site-directed Mutagenesis of GGPPs 19
2-8 Kinetic Parameter Measurements 20
2-8-1 Kinetic Constant Measurements of Km and kcat Values 20
2-8-2 Reaction Kinetics of GGPPs under Various Concentrations of Mg2+ 21
2-9 Final Products Formation and Analysis 21
2-10 Identification of the Critical Regions for Dimer Formation 22
2-10-1 Construction of N-terminal Truncated Mutants 22
2-10-2 Gel Filtration Chromatography 23
2-10-3 Circular Dichroism (CD) Experiments 23
2-11 Probing the Substrate Binding and Conformational Changes using Trp Mutants and Substrate Analog FsPP 23
2-11-1 Fluorescence Spectrophotometer Assay 24
2-12 Stopped Flow Experiments 25
2-12-1 Measurements of Substrate Binding (kon) 25
2-12-2 Monitoring the Protein Conformational Change during Catalysis 25
RESULTS 27
3-1 Purification of wild-type, SeMet-labeled, and mutats GGPPs 28
3-2 Biochemical Analysis of Recombinant S. cerevisiae GGPPs 28
3-3 Crystal Structure of S. cerevisiae GGPPs 29
3-3-1 Overall Structure 29
3-3-2 Active Site Structure 30
3-3-3 Residues Responsible for Controlling Product Chain Length 31
3-4 Mechanism of Product Chain Length Determination 31
3-4-1 Reaction Kinetics and Final Products of Different Mutants 31
3-4-2 The Molecular Ruler Mechanism 33
3-5 Mechanism of Dimer Formation of GGPPs 34
3-5-1 The Function of N-terminal Helix A 34
3-5-2 The Function of Critical Residue in Interface 35
3-6 Probing the Substrate Binding and Conformational Change 36
3-6-1 Substrates Quench the GGPPs Intrinsic Fluorescence 36
3-6-2 Comparison of Trp Mutant and Deletion Mutant in Fluorescence Assay 37
3-6-3 Stopped-Flow Experiments 37
3-7 Role of Mg2+ Ion in Catalysis 38
3-7-1 Reaction Kinetics under Different Concentration of Mg2+ 39
3-7-2 Binding Mode Probed by Fluorescence Experiments 40
DISCUSSION 41
ACKNOWLEDGMENT 48
REFERENCES 49
TABLES 56
FIGURES 65
POSTER 99
PUBLISHED PAPER 101
dc.language.isoen
dc.subject定點突變zh_TW
dc.subject一類異戊二烯轉移酵素zh_TW
dc.subject四異戊二烯焦磷酸合成酵素zh_TW
dc.subjectX光晶體繞射zh_TW
dc.subjecttrans-prenyltransferaseen
dc.subjectsite-directed mutagenesisen
dc.subjectmultiple isomorphous replacementen
dc.subjectX-ray crystallographyen
dc.subjectgeranylgeranyl pyrophosphate synthaseen
dc.title酵母菌第三型-四異戊二烯焦磷酸合成酵素之結構與功能研究zh_TW
dc.titleStructural and Functional Studies of Type-III geranylgeranyl Pyrophosphate Synthase from Saccharomyces cerevisiaeen
dc.typeThesis
dc.date.schoolyear94-2
dc.description.degree碩士
dc.contributor.oralexamcommittee王惠鈞(Andrew H.-J Wang),張文章,袁小琀(Hanna S. Yuan)
dc.subject.keyword一類異戊二烯轉移酵素,四異戊二烯焦磷酸合成酵素,X光晶體繞射,定點突變,zh_TW
dc.subject.keywordtrans-prenyltransferase,geranylgeranyl pyrophosphate synthase,X-ray crystallography,multiple isomorphous replacement,site-directed mutagenesis,en
dc.relation.page117
dc.rights.note有償授權
dc.date.accepted2006-07-21
dc.contributor.author-college生命科學院zh_TW
dc.contributor.author-dept生化科學研究所zh_TW
顯示於系所單位:生化科學研究所

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