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研究生:姚俊宇
研究生(外文):Jyun-Yu Yao
論文名稱:釀酒酵母中表現並鑑定植物順式異戊二烯轉移酶LLA66
論文名稱(外文):Expression and characterization of a plant cis-prenyltransferase LLA66 in Saccharomyces cerevisiae
指導教授:梁博煌
口試委員:楊啟伸郭致榮
口試日期:2017-06-07
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生化科學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:英文
論文頁數:65
中文關鍵詞:異戊二烯轉移酶麝香百合釀酒酵母中鏈萜類
外文關鍵詞:prenyltransferaseLilium longiflorumSaccharomyces cerevisiaemedium-chain isoprenoid
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異戊二烯轉移酶 (prenyltransferase)中有一種類的功能是催化將特定數量的異戊烯焦磷酸 (isopentenyl diphosphate, IPP)與法尼基焦磷酸 (farnesyl diphosphate, FPP)進行連續縮合反應,將碳鏈延長到指定的長度。根據受質異戊烯焦磷酸酵素反應時所形成的雙鍵立體化學,而將異戊二烯轉移酶分類為順式和反式。順式異戊二烯轉移酶主要合成較長的產物作為脂質載體,主導真核生物中糖蛋白或細菌中肽聚醣的生物合成。然而因為植物中的順式異戊烯基轉移酶通常存在幾種同源亞型,所以功能較少被仔細了解。來自麝香百合花的順式異戊烯基轉移酶基因LLA66是在絨氈層和小孢子中,所鑑定的第一個異戊烯基轉移酶。本次研究中,我們利用在釀酒酵母 (Saccharomyces cerevisiae)表現重組蛋白LLA66,發現它是一種獨特的中鏈萜類合成酶。藉由高效液相色譜法 (HPLC)及薄層色譜 (TLC)分析中,顯示產物為45個碳鏈的萜類。此外在使用Ni-NTA親和層析純化有6個His標記的LLA66蛋白時,我們還發現純化後的蛋白質溶液中具有將IPP轉化為法尼醇(farnesol)的催化活性。通過進一步的數據,我們認為是IPP:DMAPP異構酶、FPP合成酶和鹼性磷酸酶。但它們是否會形成蛋白質複合物是需要實驗的進一步驗證。
A group of prenyltransferases catalyze chain elongation of farnesyl diphosphate (FPP) to designated lengths by consecutive condensation reactions with specific numbers of isopentenyl diphosphate (IPP). According to the stereochemistry of the double bonds formed by IPP condensation, these prenyltransferase are classified as cis- and trans-types. Cis-prenyltransferases synthesize longer products as lipid carriers to mediate biosynthesis of peptidoglycan in bacteria or glycoproteins in eukaryotes. However, the functions of cis-prenyltransferases in plants are less understood because there are several homologous isoforms in different compartments. The cis-prenyltransferase gene LLA66 from Lilium longiflorum anther is the first prenyltransferase identified in the tapetum and microspores. As reported here, we have produced the recombinant LLA66 in Saccharomyces cerevisiae, performed in-vitro characterization of the enzyme and found it is a unique medium-chain synthase, and was resolved C45 isoprenoids in thin layer chromatography (TLC) and HPLC. After purification of the hexa-His-tagged LLA66 using Ni-NTA affinity chromatography, we also identified enzyme activities to convert IPP into farnesol in the purified protein mixture. By further analysis, we thought that a protein mixture of IPP:DMAPP isomerase, FPP synthase and alkaline phosphatase was co-purified with LLA66. We will further characterize the protein complex.
LITS OF TABLE
中文摘要 iv
ABSTRACT v
LITS OF TABLE vi
LIST OF FIGURE vii
ABBREVIATIONS ix
1. INTRODUCTION 1
1.1 Isoprenoid 1
1.2 Isoprenyl pyrophosphate synthases 1
1.3 In this study 2
MATERIALS AND METHODS 4
2.1 Chemicals 4
2.2 Preparation of LLA66 construct. 4
2.2.1 pYES2/CT-LLA66 construct for S.cerevisiae 5
2.2.2 LLA66 construct for E. coli 5
2.3 Extraction of polyisoprenoids from yeast 5
2.4 HPCL/UV analysis of polyisoprenoids. 6
2.5 Purification of His-tagged LLA66 7
2.5.1 Overexpression of LLA66 in S. cerevisiae 7
2.5.2 Overxpression of LLA66 in E. coli 8
2.6 Activity assay of LLA66 9
2.6.1 EnzChek Pyrophosphate assay 9
2.6.2 Radiative [14C] IPP assay 9
2.7 TLC analysis of polyisoprenoids 11
2.8 Ion exchange chromatography using Mono-S and Mono-Q columns 11
RESULTS 13
3.1 Functional characterization of LLA66 activity in S. cerevisiae rer2Δ mutant 13
3.2 HPLC/UV analysis of polyisoprenoids of Saccharomyces cerevisiae 13
3.3 LLA66 overexpression and purification in Saccharomyces cerevisiae 14
3.4 Unexpected activity of LLA66 from Saccharomyces cerevisiae expression 14
3.5 Thin layer chromatography analysis of polyisoprenoids of LLA66 15
3.6 Identification of Saccharomyces cerevisiae enzyme mixture for unexpected IPP-utilizing activity 15
3.7 Ion exchange chromatography analysis of LLA66 and yeast enzyme 17
3.8 LLA66 overexpression and purification in E. coli 17
3.9 LLA66 activity assay from E. coli expression 18
3.10 Characterization and kinetics of LLA66 enzyme 19
DISCUSSION 20
TABLES 24
FIGURES 30
REFERENCES 58
REFERENCES
1.Liang, P. H., Ko, T. P., and Wang, A. H. (2002) Structure, mechanism and function of prenyltransferases. Eur J Biochem 269, 3339-3354
2.Poulter, C. D. (1974) Model studies in terpene biosynthesis. J Agric Food Chem 22, 167-173
3.Gershenzon, J., and Dudareva, N. (2007) The function of terpene natural products in the natural world. Nat Chem Biol 3, 408-414
4.Stone, K. J., Wellburn, A. R., Hemming, F. W., and Pennock, J. F. (1967) The characterization of ficaprenol-10, -11 and -12 from the leaves of Ficus elastica (decorative rubber plant). Biochemical Journal 102, 325-330
5.Swiezewska, E., and Danikiewicz, W. (2005) Polyisoprenoids: structure, biosynthesis and function. Prog Lipid Res 44, 235-258
6.Sinensky, M. (2000) Recent advances in the study of prenylated proteins. Biochim Biophys Acta 1484, 93-106
7.Gelb, M. H., Scholten, J. D., and Sebolt-Leopold, J. S. (1998) Protein prenylation: from discovery to prospects for cancer treatment. Curr Opin Chem Biol 2, 40-48
8.Clarke, S. (1992) Protein isoprenylation and methylation at carboxyl-terminal cysteine residues. Annu Rev Biochem 61, 355-386
9.Kellogg, B. A., and Poulter, C. D. (1997) Chain elongation in the isoprenoid biosynthetic pathway. Curr Opin Chem Biol 1, 570-578
10.Ogura, K., and Koyama, T. (1998) Enzymatic Aspects of Isoprenoid Chain Elongation. Chem Rev 98, 1263-1276
11.Wang, K., and Ohnuma, S. (1999) Chain-length determination mechanism of isoprenyl diphosphate synthases and implications for molecular evolution. Trends Biochem Sci 24, 445-451
12.Schulbach, M. C., Brennan, P. J., and Crick, D. C. (2000) Identification of a short (C15) chain Z-isoprenyl diphosphate synthase and a homologous long (C50) chain isoprenyl diphosphate synthase in Mycobacterium tuberculosis. J Biol Chem 275, 22876-22881
13.Guo, R. T., Ko, T. P., Chen, A. P., Kuo, C. J., Wang, A. H., and Liang, P. H. (2005) Crystal structures of undecaprenyl pyrophosphate synthase in complex with magnesium, isopentenyl pyrophosphate, and farnesyl thiopyrophosphate: roles of the metal ion and conserved residues in catalysis. J Biol Chem 280, 20762-20774
14.Allen, C. M. (1985) Purification and characterization of undecaprenylpyrophosphate synthetase. Methods Enzymol 110, 281-299
15.Ko, T. P., Chen, Y. K., Robinson, H., Tsai, P. C., Gao, Y. G., Chen, A. P., Wang, A. H., and Liang, P. H. (2001) Mechanism of product chain length determination and the role of a flexible loop in Escherichia coli undecaprenyl-pyrophosphate synthase catalysis. J Biol Chem 276, 47474-47482
16.Bouhss, A., Trunkfield, A. E., Bugg, T. D., and Mengin-Lecreulx, D. (2008) The biosynthesis of peptidoglycan lipid-linked intermediates. FEMS Microbiol Rev 32, 208-233
17.Sato, M., Sato, K., Nishikawa, S., Hirata, A., Kato, J., and Nakano, A. (1999) The yeast RER2 gene, identified by endoplasmic reticulum protein localization mutations, encodes cis-prenyltransferase, a key enzyme in dolichol synthesis. Mol Cell Biol 19, 471-483
18.Grabinska, K., and Palamarczyk, G. (2002) Dolichol biosynthesis in the yeast Saccharomyces cerevisiae: an insight into the regulatory role of farnesyl diphosphate synthase. FEMS Yeast Res 2, 259-265
19.Chojnacki, T., and Dallner, G. (1988) The biological role of dolichol. Biochem J 251, 1-9
20.Bizzarri, R., Cerbai, B., Signori, F., Solaro, R., Bergamini, E., Tamburini, I., and Chiellini, E. (2003) New perspectives for (S)-dolichol and (S)- nor dolichol synthesis and biological functions. Biogerontology 4, 353-363
21.Jones, M. B., Rosenberg, J. N., Betenbaugh, M. J., and Krag, S. S. (2009) Structure and synthesis of polyisoprenoids used in N-glycosylation across the three domains of life. Biochim Biophys Acta 1790, 485-494
22.Oh, S. K., Han, K. H., Ryu, S. B., and Kang, H. (2000) Molecular cloning, expression, and functional analysis of a cis-prenyltransferase from Arabidopsis thaliana. Implications in rubber biosynthesis. J Biol Chem 275, 18482-18488
23.Zhang, H., Ohyama, K., Boudet, J., Chen, Z., Yang, J., Zhang, M., Muranaka, T., Maurel, C., Zhu, J. K., and Gong, Z. (2008) Dolichol biosynthesis and its effects on the unfolded protein response and abiotic stress resistance in Arabidopsis. Plant Cell 20, 1879-1898
24.Schilmiller, A. L., Schauvinhold, I., Larson, M., Xu, R., Charbonneau, A. L., Schmidt, A., Wilkerson, C., Last, R. L., and Pichersky, E. (2009) Monoterpenes in the glandular trichomes of tomato are synthesized from a neryl diphosphate precursor rather than geranyl diphosphate. Proc Natl Acad Sci U S A 106, 10865-10870
25.Akhtar, T. A., Matsuba, Y., Schauvinhold, I., Yu, G., Lees, H. A., Klein, S. E., and Pichersky, E. (2013) The tomato cis-prenyltransferase gene family. Plant J 73, 640-652
26.Hsu, Y. F., Tzeng, J. D., Liu, M. C., Yei, F. L., Chung, M. C., and Wang, C. S. (2008) Identification of anther-specific/predominant genes regulated by gibberellin during development of lily anthers. J Plant Physiol 165, 553-563
27.Tzeng, J. D., Hsu, S. W., Chung, M. C., Yeh, F. L., Yang, C. Y., Liu, M. C., Hsu, Y. F., and Wang, C. S. (2009) Expression and regulation of two novel anther-specific genes in Lilium longiflorum. J Plant Physiol 166, 417-427
28.Liu, M. C., Wang, B. J., Huang, J. K., and Wang, C. S. (2011) Expression, localization and function of a cis-prenyltransferase in the tapetum and microspores of lily anthers. Plant Cell Physiol 52, 1487-1500
29.Fujii, H., Koyama, T., and Ogura, K. (1982) Efficient enzymatic hydrolysis of polyprenyl pyrophosphates. Biochim Biophys Acta 712, 716-718
30.Kaneko, M., Itoh, H., Inukai, Y., Sakamoto, T., Ueguchi-Tanaka, M., Ashikari, M., and Matsuoka, M. (2003) Where do gibberellin biosynthesis and gibberellin signaling occur in rice plants? Plant J 35, 104-115
31.Kera, K., Takahashi, S., Sutoh, T., Koyama, T., and Nakayama, T. (2012) Identification and characterization of a cis,trans-mixed heptaprenyl diphosphate synthase from Arabidopsis thaliana. FEBS J 279, 3813-3827
32.Surmacz, L., Plochocka, D., Kania, M., Danikiewicz, W., and Swiezewska, E. (2014) cis-Prenyltransferase atCPT6 produces a family of very short-chain polyisoprenoids in planta. Biochim Biophys Acta 1841, 240-250
33.Harrison, K. D., Park, E. J., Gao, N., Kuo, A., Rush, J. S., Waechter, C. J., Lehrman, M. A., and Sessa, W. C. (2011) Nogo-B receptor is necessary for cellular dolichol biosynthesis and protein N-glycosylation. EMBO J 30, 2490-2500
34.Soding, J., Biegert, A., and Lupas, A. N. (2005) The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Res 33, W244-248
35.Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D. J. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 3389-3402
36.Zhu, W., Zhang, Y., Sinko, W., Hensler, M. E., Olson, J., Molohon, K. J., Lindert, S., Cao, R., Li, K., Wang, K., Wang, Y., Liu, Y. L., Sankovsky, A., de Oliveira, C. A., Mitchell, D. A., Nizet, V., McCammon, J. A., and Oldfield, E. (2013) Antibacterial drug leads targeting isoprenoid biosynthesis. Proc Natl Acad Sci U S A 110, 123-128
37.Creighton, T. E. (1994) The energetic ups and downs of protein folding. Nat Struct Biol 1, 135-138
38.Gibeaut, D. M., Hulett, J., Cramer, G. R., and Seemann, J. R. (1997) Maximal biomass of Arabidopsis thaliana using a simple, low-maintenance hydroponic method and favorable environmental conditions. Plant Physiology 115, 317-319
39.Arthington-Skaggs, B. A., Jradi, H., Desai, T., and Morrison, C. J. (1999) Quantitation of Ergosterol Content: Novel Method for Determination of Fluconazole Susceptibility of Candida albicans. Journal of Clinical Microbiology 37, 3332-3337
40.Van den Heuvel, K. J., Van Lipzig, R. H., Barendse, G. W., and Wullems, G. J. (2002) Regulation of expression of two novel flower-specific genes from tomato (Solanum lycopersicum) by gibberellin. J Exp Bot 53, 51-59
41.Asawatreratanakul, K., Zhang, Y. W., Wititsuwannakul, D., Wititsuwannakul, R., Takahashi, S., Rattanapittayaporn, A., and Koyama, T. (2003) Molecular cloning, expression and characterization of cDNA encoding cis-prenyltransferases from Hevea brasiliensis. A key factor participating in natural rubber biosynthesis. Eur J Biochem 270, 4671-4680
42.Skorupinska-Tudek, K., Bienkowski, T., Olszowska, O., Furmanowa, M., Chojnacki, T., Danikiewicz, W., and Swiezewska, E. (2003) Divergent pattern of polyisoprenoid alcohols in the tissues of Coluria geoides: a new electrospray ionization MS approach. Lipids 38, 981-990
43.Westbrook, J., Feng, Z., Chen, L., Yang, H., and Berman, H. M. (2003) The Protein Data Bank and structural genomics. Nucleic Acids Research 31, 489-491
44.Samuelson, J., Banerjee, S., Magnelli, P., Cui, J., Kelleher, D. J., Gilmore, R., and Robbins, P. W. (2005) The diversity of dolichol-linked precursors to Asn-linked glycans likely results from secondary loss of sets of glycosyltransferases. Proc Natl Acad Sci U S A 102, 1548-1553
45.Sanders, J. M., Song, Y., Chan, J. M. W., Zhang, Y., Jennings, S., Kosztowski, T., Odeh, S., Flessner, R., Schwerdtfeger, C., Kotsikorou, E., Meints, G. A., Gómez, A. O., González-Pacanowska, D., Raker, A. M., Wang, H., van Beek, E. R., Papapoulos, S. E., Morita, C. T., and Oldfield, E. (2005) Pyridinium-1-yl Bisphosphonates Are Potent Inhibitors of Farnesyl Diphosphate Synthase and Bone Resorption. Journal of Medicinal Chemistry 48, 2957-2963
46.Bajda, A., Konopka-Postupolska, D., Krzymowska, M., Hennig, J., Skorupinska-Tudek, K., Surmacz, L., Wojcik, J., Matysiak, Z., Chojnacki, T., Skorzynska-Polit, E., Drazkiewicz, M., Patrzylas, P., Tomaszewska, M., Kania, M., Swist, M., Danikiewicz, W., Piotrowska, W., and Swiezewska, E. (2009) Role of polyisoprenoids in tobacco resistance against biotic stresses. Physiol Plant 135, 351-364
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