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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 林俊宏 | |
dc.contributor.author | Tzann-Shun Hwang | en |
dc.contributor.author | 黃贊勳 | zh_TW |
dc.date.accessioned | 2021-06-08T05:20:03Z | - |
dc.date.copyright | 2005-07-30 | |
dc.date.issued | 2005 | |
dc.date.submitted | 2005-07-27 | |
dc.identifier.citation | Annunziato, P.W., Wright, L.F., Vann, W.F. and Silver, R.P. (1995) Nucleotide sequence and genetic analysis of the neuD and neuB genes in region 2 of the polysialic acid gene cluster of Escherichia coli K1. J. Bacteriol. 177, 312-319.
Anton, D.L., Hedstrom, L. Fish, S.M. and Abeles, R.H. (1983) Mechanism of enolpyruvyl shikimate-3-phosphate synthase exchange of phosphoenolpyruvate with solvent protons. Biochemistry 22, 5093-5908. Blacklow, R.S. and Warren, L. (1962) Biosynthesis of sialic acids by Neisseria meningitides. J. Biol. Chem. 237, 3520-3526. Bravo, I.G., Garcia-Vallve, S., Romeu, A. and Reglero, A. (2004) Prokaryotic origin of cytidylyltransferases and alpha-ketoacid synthases. Trends Microbiol. 12, 120-128. Chen, H., Blume, A., Zimmermann-Kordmann, M., Reutter, W. and Hinderlich, S. (2002) Purification and characterization of N-acetylneuraminic acid-9-phosphate synthase from rat liver. Glycobiology 12, 65-71. Dotson, G.D., Nanjappan, P., Reily, M.D. and Woodard, R.W. (1993) Stereochemistry of 3-deoxyoctulosonate 8-phosphate synthase Biochemistry 32, 12392-12397. Duewel, H.S., Radaev, S., Wang, J., Woodard, R.W. and Gatti, D.L. (2001) Substrate and metal complexes of 3-deoxy-D-manno-octulosonate-8-phosphate synthase from Aquifex aeolicus at 1.9-A resolution. Implications for the condensation mechanism. J. Biol. Chem. 276, 8393-8402. Edwards, M.S., Kasper, D.L., Jennings, H.J., Baker, C.J. and Nicholson-Weller, A. (1982) Capsular sialic acid prevents activation of the alternative complement pathway by type III, group B streptococci. J. Immunol. 128, 1278-1283. Glaser, P., Rusniok, C., Buchrieser, C., Chevalier, F., Frangeul, L., Msadek, T., Zouine, M., Couve, E., Lalioui, L., Poyart, C., Trieu-Cuot, P. and Kunst, F. (2002) Genome sequence of Streptococcus agalactiae, a pathogen causing invasive neonatal disease. Mol. Microbiol. 45, 1499-1513. Gotschlich, E.C., Fraser, B.A., Nishimura, O., Robbins, J.B. and Liu, T.Y. (1981) Lipid on capsular polysaccharides of Gram- negative bacteria. J. Biol. Chem. 256, 8915-8921. Gunawan, J., Simard, D., Gilbert, M., Lovering, A.L., Wakarchuk, W.W., Tanner, M.E., Strynadka, N.C. (2005) Structural and mechanistic analysis of sialic acid synthase NeuB from Neisseria meningitidis in complex with Mn2+, phosphoenolpyruvate, and N-acetylmannosaminitol. J. Biol. Chem. 280, 3555-3563 Hedstrom, L. and Abeles, R. (1988) 3-Deoxy-D-manno-octulosonate-8-phosphate synthase catalyzes the C-O bond cleavage of phosphoenolpyruvate. Biochem. Biophys. Res. Commun. 157, 816-820. Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K., Pease,L.R. (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction, Gene 77, 51– 59. Howe, D.L., Sundaram, A.K., Wu, J., Gatti, D.L., and Woodard, R.W. (2003) Mechanistic insight into 3-deoxy-D-manno-octulosonate-8-phosphate synthase and 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase utilizing phosphorylated monosaccharide analogues. Biochemistry 42, 4843-4854. Izard, T., Lawrence, M.C., Malby, R.L., Lilley, G.G. and Colman, P.M. (1994) The three-dimensional structure of N-aceytlneuraminate lyase from E. coli. Structure 2, 361-369. Jennings, H.J., Katzenellenbogen, E., Lugowski, C., Michon, F., Roy, R., and Kaspar, D.L. (1984) Structure, conformationand immunology of sialic acid-containing polysaccharides of human pathogenic bacteria. Pure Appl. Chem. 56, 893-905. Kaijser, B., Hanson,L.A., Jodal,V., Lindin-Janson, G. and Robbins, J.B. (1977) Frequency of E.coli K antigens in urinary tract infections in children. Lancet 1, 664-666. Kelm, S. and Schauer, R. (1997) Sialic Acid in Molecular and Cellular Interactions. Int. Rev. Cytol. 175, 137-240. Kim, K., Lawrence, S.M., Park, J., Pitts, L., Vann, W.F., Betenbaugh, M.J. and Palter, K.B. (2002) Expression of a functional Drosophila melanogaster N-acetylneuraminic acid (Neu5Ac) phosphate synthase gene: evidence for endogenous sialic acid biosynthetic ability in insects. Glycobiology 12, 73-83. Kohen, A., Berkovich, R., Belakhov, V. and Baasov T. (1993) Stereochemistry of the KDO8P synthase. An efficient synthesis of the 3-fluoro analogues of KDO8P. Bioorg. Med. Chem. Lett. 3, 1577-1582. Kohen, A., Jakob, A., and Baasov, T. (1992) Mechanistic Studies of 3-deoxy-D-manno-2-octulosonate-8 phosphate synthase from Escherichia coli. Eur. J. Biochem. 208, 443- Komaki, E. Ohta, Y. and Tsudada, Y. (1997) Purification and characterization of N-aceytlneuraminate synthase from Escherichia coli K1-M12, Biosci. Biotech. Biochem. 61, 2046-2050. Krekel, F., Oecking, C., Amrhein, N., Macheroux, P. (1999) Substrate and inhibitor-induced conformational changes in the structurally related enzymes UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) and 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS). Biochemistry 38, 8864-8878 Lawrence, S.M., Huddleston, K.A., Pitts, L.R., Nguyen, N., Lee, Y.C., Vann, W.F., Coleman, T.A. and Betenbaugh, M.J. (2000) Cloning and expression of the human N-acetylneuraminic acid phosphate synthase gene with 2-keto-3-deoxy-D-glycero- D-galacto-nononic acid biosynthetic ability. J. Biol. Chem. 275, 17869-17877. Lebioda, L., Stec, B., Brewer, J.M. and Tykarska, E. (1991) Inhibition of enolase: the crystal structures of enolase-Ca2+-2-phosphoglycerate and enolase-Zn2+-phosphoglycolate complexes at 2.2-A resolution. Biochemistry 30, 2823-2827. Lewis, A.L., Nizet, V. and Varki, A. (2004) Discovery and characterization of sialic acid O-acetylation in group B Streptococcus. Proc. Natl. Acad. Sci. USA 101, 11123-11128. Liang, P.H., Lewis J., and Anderson K.S. (1998) Catalytic mechanism of KDO8P synthase: transient kinetic studies and evaluation of a putative reaction intermediate. Biochemistry 37, 16390-16399 Linton, D., Karlyshev, A.V., Hitchen, P.G., Morris, H.R., Dell, A., Gregson, N.A. and Wren, B.W. (2000) Multiple N-acetyl neuraminic acid synthetase (neuB) genes in Campylobacter jejuni: identification and characterization of the gene involved in sialylation of lipo-oligosaccharide. Mol. Microbiol. 35, 1120-1134. Lipari, F. and Herscovics, A. (1996) Role of the cysteine residues in the alpha1,2-mannosidase involved in N-glycan biosynthesis in Saccharomyces cerevisiae. The conserved Cys340 and Cys385 residues form an essential disulfide bond. J. Biol. Chem. 271, 27615-27622. Lolis, E. and Petsko, G.A. (1990) Crystallographic analysis of the complex between triosephosphate isomerase and 2-phosphoglycolate at 2.5-A resolution: implications for catalysis. Biochemistry 29, 6619-6625. Marquardt, J.L., Brown, E.D., Lane, W.S., Haley, T.M., Ichikawa, Y., Wong, C.H. and Walsh, C.T. (1994) Kinetics, stoichiometry, and identification of the reactive thiolate in the inactivation of UDP-GlcNAc enolpyruvoyl transferase by the antibiotic fosfomycin. Biochemistry 33, 10646-10651. Marques, M.B., Kasper, D.L., Pangburn, M.K., and Wessels, M.R. (1992) Prevention of C3 deposition by capsular polysaccharide is a virulence mechanism of type III group streptococci. Infect. Immun. 60, 3986-3993. Meloche, H.P. (1967) Bromopyruvate inactivation of 2-keto-3-deoxy-6-phosphogluconic aldolase. I. Kinetic evidence for active site specificity. Biochemistry 6, 2273-2280. Mitchell, T.J. (2003) The pathogenesis of streptococcal infections: from tooth decay to meningitis. Nat. Rev. Microbiol. 1, 219-230. Nakata, D., Close, B.E., Colley, K.J., Matsuda, T., and Kitajima, K. (2000) Molecular cloning and expression of the mouse N-acetylneuraminic acid 9-phosphate synthase which does not have deaminoneuraminic acid (KDN) 9-phosphate synthase activity. Biochem. Biophys. Res. Commun. 273, 642-648. Pace, C.N., Vajdos, F., Fee, L., Grimsley, G. and Gray, T. (1995) How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 4, 2411-2423. Pudota, B.N., Miyagi, M., Hallgren, K.W., West, K.A., Crabb, J.W., Misono, K.S., and Berkner, K.L . (2000) Identification of the vitamin K-dependent carboxylase active site: Cys-99 and Cys-450 are required for both epoxidation and carboxylation. Proc. Natl. Acad. Sci. USA 97, 13033-13308. Radaev, S., Dastidar, P., Patel. M., Woodard, R.W. and Gatti, D.L. (2000) Structure and mechanism of 3-deoxy-D-nanno-octulosonate 8-phosphate synthase. J. Biol. Chem. 275, 9476-9484. Ray, J.M. and Bauerle, R. (1991) Purification and properties of tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli. J. Bacteriol. 173, 1894-1901 Salleh, H.M., Patel, M.A. and Woodard, R.W. (1996) Essential cysteines in 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase from Escherichia coli: analysis by chemical modification and site-directed mutagenesis. Biochemistry 35, 8942-8947. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2th Edition. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Schauer, R., Kelm, S., Reuter, G., Roggentin, P., and Shaw, L. (1995) Biochemistry and role of sialic acids. In Rosenberg, A. (ed.) Biology of the sialic acids. New York. pp. 7-67. Schonbrunn, E., Eschenburg, S., Shuttleworth, W.A., Schloss, J.V., Amrhein, N., Evans, J.N. and Kabsch, W. (2001) Interaction of the herbicide glyphosate with its target enzyme 5-enolpyruvylshikimate 3-phosphate synthase in atomic detail. Proc. Natl. Acad. Sci. U.S.A. 98, 1376-1380. Schonbrunn, E., Sack, S., Eschenburg, S., Perrakis, A., Krekel, F., Amrhein, N. and Mandelkow, E. (1996) Crystal structure of UDP-N-acetylglucosamine enolpyruvyltransferase, the target of the antibiotic fosfomycin. Structure 4, 1065-1075. Schoner, R., and Herrmann, K.M. (1976) 3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase. Purification, properties, and kinetics of the tyrosine-sensitive isoenzyme from Escherichia coli. J. Biol. Chem. 251, 5440-5447. Shumilin, I.A., Kretsinger, R.H., and Bauerle, R.H. (1999) Crystal structure of phenylalanine-regulated 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli. Structure 7, 865-875. Silver, R.P., Finn, C.V., Vann, W.F., Aaronson, W., Schneerson, R., Kretschmer, P.J. and Caron, C.F. (1981) Molecular cloning of the K1 capsular polysaccharide genes of E. coli. Nature 289, 696-698. Silver, R.P., Vann, W.F. and Aaronson, W. (1984) Genetic and molecular analysis of Escherichia coli K1 antigen genes. J. Bacteriol 157, 568-575. Skarzynski, T., Kim, D.H., Lees, W.J., Walsh, C.T. and Duncan, K. (1998) Stereochemical course of enzymatic enolpyruvyl transfer and catalytic conformation of the active site revealed by the crystal structure of the fluorinated analogue of the reaction tetrahedral intermediate bound to the active site of the C115A mutant of MurA. Biochemistry 37, 2572-2577 Sreerama, N. and Woody, R.W. (1993) A self-consistent method for the analysis of protein secondary structure from circular dichroism. Anal. Biochem. 209, 32-44. Sreerama, N. and Woody, R.W. (1994) Poly(pro)II helices in globular proteins: identification and circular dichroic analysis. Biochemistry 33, 10022-10025. Staub, M. and Denes, G. (1969) Purification and properties of the 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (phenylalanine sensitive) of Escherichia coli K12. I. Purification of enzyme and some of its catalytic properties. Biochim Biophys Acta. 27, 588-98. Stephens, C.M. and Bauerle, R. (1992) Essential cysteines in 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli. Analysis by chemical modification and site-directed mutagenesis of the phenylalanine-sensitive isozyme. J. Biol. Chem. 267, 5762-5767. Sundaram, A.K., Pitts, L., Muhammad, K., Wu, J., Betenbaugh, M., Woodard, R.W., and Vann, W.F. (2004) Characterization of N-acetylneuraminic acid synthase isoenzyme 1 from Campylobacter jejuni. Biochem. J. 383, 83-89. Suryaniti, V., Nelson, A. and Berry, A. (2003) Cloning, over-expression, purification, and characterization of N-acetylneuraminate synthase from Streptococcus agalactiae. Protein Expr. Purif. 27, 346-356 Takahashi, S., Aoyagi, Y., Adderson, E.E., Okuwaki, Y., and Bohnsack, J.F. (1999) Capsular sialic acid limits C5a production on type III group B streptococci. Infect. Immun. 67, 1866-1870. Tettelin, H., Masignani, V., Cieslewicz, M.J., Eisen, J.A., Peterson, S., Wessels, M.R., Paulsen, I.T., Nelson, K.E., Margarit, I., Read, T.D., Madoff, L.C., Wolf, A.M., Beanan, M.J., Brinkac, L.M., Daugherty, S.C., DeBoy, R.T., Durkin, A.S., Kolonay, J.F., Madupu, R., Lewis, M.R., Radune, D., Fedorova, N.B., Scanlan, D., Khouri, H., Mulligan, S., Carty, H.A., Cline, R.T., Van Aken, S.E., Gill, J., Scarselli, M., Mora, M., Iacobini, E.T., Brettoni, C., Galli, G., Mariani, M., Vegni, F., Maione, D., Rinaudo, D., Rappuoli, R., Telford, J.L., Kasper, D.L., Grandi, G. and Fraser, C.M. (2002) Complete genome sequence and comparative genomic analysis of an emerging human pathogen, serotype V Streptococcus agalactiae. Proc. Natl. Acad. Sci. USA. 99, 12391-12396. Vann, W.F., Tavarez, J.J., Crowley, J., Vimr, E., and Silver, R.P. (1997) Purification and characterization of the Escherichia coli K1 neuB gene product N-aceylneuraminic acid synthetase. Glycobiology 7, 697-701. Varki, A. (1992) Diversity in the sialic acids. Glycobiology 2, 24-40. Vimr, E.R., and Aaronson, W. and Silver, R.P. (1989) Genietic analysis of chromosomal mutations in the polysialic acid gene cluster of Escherichia coli K1. J. Bacteriol. 171, 1106-1117. Wagner, T., Shumilin, I.A., Bauerle, R. and Kretsinger, R.H. (2000) Structure of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase from Escherichia coli: comparison of the Mn(2+)*2-phosphoglycolate and the Pb(2+)*2-phosphoenolpyruvate complexes and implications for catalysis. J. Mol. Biol. 301, 389-399. Warren, L. (1959) The thiobarbituric acid assay of sialic acids. J. Biol. Chem. 234, 1971-1975. Watson, R.G. and Scherp, H.W. (1958a) The specific hapten of group C (group II alpha) meningococcus. I. Preparation and immunological behavior. J. Immunol. 81, 331-337. Watson, R.G., Marinetti, G.V. and Scherp, H.W. (1958b) The specific hapten of group C (group II alpha) meningococcus. II. Chemical nature. J. Immunol. 81, 337. Wessels, M.R., Benedi, V.-J., Jennings, H.J., Mechon, F., DiFabio, J.L., and Kaspar, D.L. (1989a) Isolation and characterization of type IV group B streptococcus capsular polysaccharide. Infect. Immun. 57, 1089-1094. Wessels, M.R., DiFabio, J.L., Benedi, V.-J., Kasper D.L., Michon, F., Brisson, J.R., Jelinkova, J., and Jennings, H.J. (1991) Structural determination and immunochemical characterization of type V group B streptococcus capsular polysaccharide. J. Biol. Chem. 266, 6714-6719. Wessels, M.R., Rubens, C.E., Benedi, V.-J., and Kaspar, D.L. (1989b) Definition of a bacterial virulence factor: Sialylation of the group B streptococcal capsule. Proc. Natl. Acad. Sci. USA. 86, 8983-8987. Wilkinson, H.W. (1978) Group B streptococcal infection in humans. Ann. Rev. Microbiol. 32, 41-57. 陳嘉元, 台灣大學理學院生化科學研究所碩士論文 (2000). 選殖與大量表現大腸桿菌K1之N-乙醯神經胺糖酸合成酶及其酵素特性分析. 張瓊方, 台灣大學理學院生化科學研究所碩士論文 (2003). 以定點突變方式探討大腸桿菌K1唾液酸合成酶之活性區. 陳瑞娟, 台灣大學生命科學院生化科學研究所碩士論文 (2004). 以化學修飾法與定點突變法研究無乳鏈球菌之唾液酸合成酶內必需的半胱胺酸與精胺酸. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/24262 | - |
dc.description.abstract | 大腸桿菌K1可導致新生兒腦膜炎,其菌體表面的莢膜含有alpha2,8鍵結的聚唾液酸,是感染寄主細胞的致病因子。無乳糖鏈球菌的莢膜含有alpha2,3鍵結的唾液酸,能抑制補體C3b的作用及防止巨噬細胞的吞噬,造成新生兒敗血症或腦膜炎。此兩菌株負責唾液酸合成的基因都是位於kps 基因叢的neuB,其基因產物為唾液酸合成酶 (EC 4.1.3.19, NeuB),可催化ManNAc與PEP的縮合反應,生成唾液酸。此酵素的產物與病源菌的致病性相關,因此對酵素活性區的探討,有助於藥物的開發,以及此酵素在唾液酸合成的應用。
本研究將大腸桿菌K1的唾液酸合成酶 (EcNeuB) 和無乳糖鏈球菌的唾液酸合成酶 (SaNeuB) 從基因體DNA中選殖出來,建立酵素的最適反應條件,包括最適酸鹼度、溫度、穩定度和金屬離子的需求等特性,並且進行生化特性的探討。EcNeuB在純化時,發現在Lys280處會被蛋白質水解酶切割,使原本約40 kDa的蛋白質,斷裂成33 kDa及7 kDa兩個片段,造成酵素失去活性。以MALDI-TOF-MS和化學鏈結法進行分析,顯示斷裂後的蛋白質會由原本的四聚體變成三聚體,以圓二極光譜儀分析40 kDa及33 kDa的二級結構,發現斷裂會造成33 kDa蛋白質alpha-helix比例的減少及beta-sheet比例的增加。SaNeuB以化學鏈結法分析也呈現四聚體的結構。以nano-spray ESI-MS及電子顯微鏡對EcNeuB和SaNeuB的四級結構做進一步的探討,發現這兩個酵素是以二聚體二聚體的交互作用形成的四聚體結構。 利用專一性化學修飾法及定點突變法,針對活性區內可能參與金屬與PEP結合的半胱胺酸殘基(Cys)進行探討。Cys的修飾試劑作用可完全抑制EcNeuB和SaNeuB的活性,並且活性會隨試劑作用的時間及濃度增加而降低。DTNB滴定分析顯示EcNeuB和SaNeuB皆沒有雙硫鍵的存在,證明修飾試劑造成酵素失去活性,是因為酵素失去重要的Cys,而與雙硫鍵的破壞無關。受質保護實驗顯示PEP+Mn2+可保護酵素不被IAA或BrPy作用,使酵素維持一定的活性,證實重要的Cys是位於活性區中,而且與PEP及Mn2+的結合有關。定點突變法、酵素活性及動力學分析顯示EcNeuB的突變株C12A和C176A與SaNeuB的突變株C10A和C169A的活性降低,且對受質PEP+Mn2+的親合力也明顯降低,但在所有定點突變株與野生型的圓二極光譜分析上並沒有顯著的差異,顯示EcNeuB的Cys12和Cys176和SaNeuB的Cys10和Cys169是與受質PEP及Mn2+結合有關。以BrPy標誌的SaNeuB進行質譜分析,結果發現被標示的Cys出現在含有Cys10和Cys169的胜肽片段上,再次證實Cys10和Cys169確實位於活性區內。以腦膜炎雙球菌唾液酸合成酶的晶體結構進行SaNeuB結構的分子模擬,以瞭解Cys10,Cys169在空間中的位置及可能扮演的功能與角色。模擬的SaNeuB結構顯示Cys169會和受質PEP的磷酸基產生交互作用,與受質PEP的結合有關,Cys10則被發現會和Gln37產生交互作用,間接影響酵素對受質 PEP的結合能力。 | zh_TW |
dc.description.abstract | The capsular polysaccharide of Escherichia coli K1 containing alpha2,8-linked polysialic acid has been recognized as an important virulent determinant to cause invasive infections such as neonatal meningitis and septicemia. The capsular polysaccharide of Streptococcus agalactiae (Group B Streptococci, GBS) containing alpha2,3-sialylated oligosaccharide is also a critical virulence factor for causing neonatal septicemia, pyogenic meningitis and pneumonia. The sialic acid presented in GBS capsule has been found enhancing resistance to phagocytosis by inhibiting the alternative pathway of complement cascade to evade host defenses. In E. coli and S. agalactiae, the gene responsible for the biosynthesis of sialic acid is the neuB gene in kps gene cluster. Sialic acid synthase (NeuB), encoded by neuB gene, can catalyze the condensing reaction of N-acetylmannosamine (ManNAc) and phosphoenolpyruvate (PEP) to form N-acetylneuraminic acid (NeuAc). The product of NeuB is relative to the infection of pathogens; therefore, the investigation on the active site of NeuB is a good approach for drug design and enzyme’s application.
In this study, neuB genes from E. coli and S. agalactiae were cloned from genomic DNA and over-expressed as EcNeuB and SaNeuB, respectively. Optimal conditions for enzyme reaction, including pH, temperature, stability and metal requirement, were established. Characterization of EcNeuB and SaNeuB was also conducted. In the preparation of EcNeuB, a specific cleavage by endogenous protease(s) was found at Lys280 of sialic acid synthase (40 kDa). The cleavage results in the formation of two inactive fragments of 33 kDa and 7 kDa. The CD, MALDI-TOF-MS and chemical cross-linking studies demonstrated that the fragmentation is associated with a significant change of the enzyme from a tetrameric to trimeric form. Further studies by nano-spray ESI-MS and electron microscopy demonstrated NeuB existed in a tetrameric form by dimer-dimer interaction. Sulfhydryl-modifying reagents were able to completely inactivate the enzyme activity. The iodoacetic acid (IAA) inactivation of EcNeuB and SaNeuB was in a time- and dose-dependent manner, which revealed that Cys was important for enzyme activity. Study of the sulfhydryl group by 5,5-dithiobis-2-nitrobenzoate (DTNB) titration showed no disulfide bond in both EcNeuB and SaNeuB, suggesting the activity loss was caused by the modification of Cys residue. Site-directed mutagenesis, enzyme assay and kinetic analysis showed that C12A and C176A of EcNeuB and C10A and C169A of SaNeuB were important for enzyme activity. The substrate protection experiments indicated that aforementioned Cys residues were located in the active site and involved in the binding of PEP and Mn2+, since the substrate binding could prevent NeuB from the inactivation of IAA and bromopyruvate. Further studies on substrate protection of SaNeuB mutants and molecular modeling of SaNeuB showed that Cys169 was a residue involved in the binding of PEP and Mn2+. On the other hand, Cys10 was proposed to interact with Gln37 that is essential for the binding of PEP. Chemical modification and site-directed mutagenesis showed Arg301 and Arg277 of SaNeuB were essential in the substrate binding. Molecular modeling of SaNeuB showed that Arg301 and Arg277 were involved in the binding of ManNAc and PEP, respectively. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T05:20:03Z (GMT). No. of bitstreams: 1 ntu-94-D88242004-1.pdf: 3437772 bytes, checksum: 16a02095935c5d91a4552ea24e0ffe86 (MD5) Previous issue date: 2005 | en |
dc.description.tableofcontents | 目錄 I
圖表目次 V 縮寫表 X 中文摘要 XI 英文摘要 XII 壹. 背景介紹 1 一.唾液酸 (sialic acid) 1 1.1唾液酸的結構與唾液酸醣鏈分子 1 1.2 唾液酸的功能 2 1.3 唾液酸的合成與代謝 2 二.唾液酸對病原菌的重要性 3 2.1 唾液酸在無乳鏈球菌 (Streptococcus agalactiae) 的角色 4 2.2 唾液酸在大腸桿菌 (Escherichia coli) 的角色 4 三.唾液酸合成酶的研究概況 5 3.1 大腸桿菌K1 (Escherichia coli K1) 唾液酸合成酶 6 3.2 無乳鏈球菌 (Streptococcus agalactiae) 唾液酸合成酶 7 3.3 曲狀桿菌 (Campylobacter jejuni) 唾液酸合成酶 8 3.4 腦膜炎雙球菌 (Neisseria menigitidis) 唾液酸合成酶 8 3.5 果蠅 (Drosophila malanogaster) 唾液酸合成酶 9 3.6 小鼠 (Mouse) 唾液酸合成酶 9 3.7 大鼠 (Rat) 唾液酸合成酶 10 3.8 人類 (Human) 唾液酸合成酶 10 3.9 唾液酸合成酶一般特性及動力學分析比較 11 四.唾液酸合成酶及受質為PEP的相關酵素對活性區 (active site) 的研究 11 4.1 唾液酸合成酶 12 4.2 KDO8P 合成酶 14 4.3 DAH7P 合成酶 16 4.4 EPSP合成酶 17 4.5 UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) 17 4.6 受質為PEP的相關酵素中對Cys 殘基的研究 18 4.7 受質為PEP的相關酵素中對Arg 殘基的研究 21 五.實驗動機 22 貳. 材料與方法 23 一.實驗材料 23 1.1 菌株及載體 23 1.2 試藥及實驗相關酵素 23 1.3 儀器設備 23 二.實驗方法 23 2.1 染色體抽取 24 2.2 基因選殖 24 2.3 兩步驟聚合酶連鎖反應 (two step PCR) 建構定點突變基因 27 2.4 蛋白質的純化 27 2.5 蛋白質定量-Bradford法及吸光法定量法 29 2.6 蛋白質聚丙醯胺膠體電泳 (SDS-PAGE) 之分析 30 2.7 西方墨點法(Western hybridization) 31 2.8 等電點電泳 32 2.9 蛋白質鑑定 32 三. 酵素活性與特性的分析 32 3.1唾液酸的定量分析-TBA (thiobarbituric acid) assay 32 3.2 酵素比活性測定 33 3.3 酵素動力學測定 34 3.4 圓二極光譜 (Circular Dichroism, CD)分析 34 3.5 半胱胺酸化學修飾試劑的抑制實驗 35 3.6 不同濃度之IAA於不同時間的抑制實驗 35 3.7 受質保護實驗 (Substrate protection test) 36 3.8 DTNB滴定試驗 (DTNB titration) 36 3.9超高速離心沉降分析 36 參. 實驗結果與討論 39 一. 唾液酸合成酶基因的選殖、蛋白質的純化及酵素反應條件的建立 39 1.1 唾液酸合成酶基因的選殖 39 1.2 唾液酸合成酶的純化與活性分析 40 1.3 建立唾液酸合成酶的最適反應條件 42 1.4 二價金屬離子對唾液酸合成酶活性的影響 43 二.唾液酸合成酶的分子結構定性分析 45 2.1 唾液酸合成酶的斷裂現象及斷裂對活性的影響 45 2.2 唾液酸合成酶的斷裂對結構的影響 46 2.3 EcNeuB 和 SaNeuB的二級結構及四級結構的分析 48 2.4 酸鹼度對四級結構的影響的分析 49 2.5 電子顯微鏡對唾液酸合成酶的分析及四級結構的重建 49 三.唾液酸合成酶的生物化學及生物物理特性分析 51 3.1唾液酸合成酶的等電點分析 51 3.2 二價金屬在唾液酸合成酶中的計量分析 51 3.3 二價金屬離子濃度及鹽離子濃度對活性的效應與二級結構的分析 52 3.4 二價金屬對唾液酸合成酶穩定性的探討 52 3.5 長期保存條件的探討 53 3.6 受質特異性分析 53 3.7 動力學參數分析 54 3.8 催化機制的分析 55 3.9 受質類似物的抑制效果的分析 55 四. 半胱胺酸殘基在唾液酸合成酶中的角色 56 4.1硫氫基團修飾試劑對唾液酸合成酶的活性抑制作用 56 4.2唾液酸合成酶突變株的動力學參數分析 57 4.3 DTNB滴定法定量雙硫鍵數目 58 4.4受質保護試驗的探討 60 4.5 BrPy對唾液酸合成酶活性抑制的分析 61 4.6液相層析-質譜法分析唾液酸合成酶中被標誌的半胱胺酸 62 4.7 SaNeuB的分子模擬結構對半胱胺酸殘基角色的解釋 63 五. 精胺酸殘基在唾液酸合成酶中的角色 64 5.1 唾液酸合成酶精胺酸殘基的化學修飾、定點突變與受質保護試驗的研究 64 5.2唾酸合成酶的精胺酸突變株的活性分析 65 5.3 SaNeuB的分子模擬結構對精胺酸殘基角色的解釋 65 肆. 綜合討論 67 伍. 參考文獻 71 陸. 圖表 78 柒. 附錄 167 | |
dc.language.iso | zh-TW | |
dc.title | 唾液酸合成酶:結構分析與鑑定參與催化反應的胺基酸殘基 | zh_TW |
dc.title | Sialic Acid Synthase from Escherichia coli and Streptococcus agalactiae: Structural Characterization and Identification of Essential Catalytic Residues | en |
dc.type | Thesis | |
dc.date.schoolyear | 93-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 張固剛,張文章,廖淑惠,陳玉如,邱式鴻 | |
dc.subject.keyword | 唾液酸合成酶, | zh_TW |
dc.subject.keyword | Sialic Acid Synthase,Escherichia coli,Streptococcus agalactiae, | en |
dc.relation.page | 187 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2005-07-28 | |
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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