( 您好!臺灣時間:2021/07/24 18:13
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::


研究生(外文):Yu-Jui Chang
論文名稱(外文):Cloning, Isolation, Characterization, Crystallization and Structural Determination of Bacillus subtilis Guanine Deaminase
指導教授(外文):Shwu-Huey LiawZo-Nan Chang
外文關鍵詞:Guanine Deaminasecytidine deaminase superfamilyStructural Determinationdomain swapping
  • 被引用被引用:4
  • 點閱點閱:97
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
Guanine Deaminase (GD, EC 可對guanine進行去胺反應,並將之催化成xanthine,是核酸新陳代謝的重要酵素之一。本篇論文先由大腸桿菌表現系統得到高純度的Bacillus subtilis GD (bGD) 重組蛋白,進行酵素活性分析後得知其Km和kcat分別為56 μM和20.8 s-1。接著成功地培養出bGD蛋白晶體,以X光照射晶體後得到繞射點,利用Se-MAD的方法測定出蛋白結構,解析度為1.15 Å。本結構屬於cytidine deaminase (CDA) fold,其較特殊處是兩單體的domain-swapped C端,由helices αD1與αD2延伸至對方的活性區,且其末端的殘基 (Tyr 156) 直接與受質作用,使兩單體互相纏繞而形成緊密的二聚體結構,以3900 Å2的面積互相接觸。在無外加任何金屬離子的情形下,仍有一鋅離子緊密結合於活性區,與His53,Cys83,Cys86和一個水分子形成四面體鍵結 (tetrahedral coordination)。根據活性區所結合的imidazole和水分子,可模擬反應中間產物與bGD之交互作用,以說明bGD之催化機制和受質的辨識。此晶體結構為封閉構型 (closed conformation),顯示了當受質進入活性區時,C端尾部會與受質作用,因而覆蓋住活性區。所以對於bGD而言,C端的domain swapping不但可幫助二聚體的形成並提升結構的穩定度,而且還可誘導辨識受質的殘基在正確的空間位置,以達受質特異性。與其他CDA superfamily成員的結構比較,顯示了這些成員結構之相同與相異處,蛋白結構的可塑性雕塑了蛋白功能的多樣性。這些結構的比較,幫助了我們預測此超家族尚未解出結構之蛋白成員,其可能參與受質辨識之殘基。
Guanine deaminase (GD), a key enzyme in the nucleotide metabolism, catalyzes the hydrolytic deamination of guanine into xanthine. In this thesis, the 156-residue GD from Bacillus subtilis was first cloned, expressed and isolated. The Km and kcat value is 56 μM and 20.8 s-1. The crystal structure has been solved at 1.15 Å resolution by the Se-MAD method. Unexpectedly, the C-terminal segment is swapped to form an inter-subunit active site and an intertwined dimer with an extensive interface of 3900 Å2 per monomer. The essential zinc ion is ligated by a water molecule together with His53, Cys83 and Cys86. The reaction intermediate was modeled into the active-site cavity based on the tightly bound imidazole and water molecules, showing the conserved deamination mechanism and the specific substrate recognition by Asp114 and Tyr156’. The closed conformation reveals that substrate binding seals the active-site entrance, which is controlled by the C-terminal tail. Therefore, the domain swapping in GD contributes not only for the oligomerization, the structural stability, but also for the substrate specificity. Finally, structural comparison of the cytidine deaminase superfamily illustrates the functional versatility of the divergent active sites in guanine, cytosine and cytidine deaminases, and suggests putative specific substrate-interacting residues for other members such as the dCMP deaminase.
Bennett, M. J., Choe, S., and Eisenberg, D. (1994). "Refined structure of dimeric diphtheria toxin at 2.0 Å resolution." Protein Sci. 3, 1444-1463.
Berger, S. J., Carter, J. G., and Lowry, O. H. (1985). "Distribution of guanine deaminase in mouse brain." J. Neurochem. 44, 1736-1740.
Betts, L., Xiang, S. S., Short, A., Wolfenden, R., and Carter, C. W. Jr. (1994). "Cytidine deaminase. The 2.3 Å crystal structure of an enzyme: transition-state analog complex." J. Mol. Biol. 235, 635-656.
Brosh, S., Sperling, O., Bromberg, Y., and Sidi, Y. (1990). "Developmental changes in the activity of enzymes of purine metabolism in rat neuronal cells in culture and in whole brain." J. Neurochem. 54, 1776-1781.
Canbolat, O., Cetin, I. R., Kavutcu, M. S., Demirci and Ozturk, S. (1996). "Activities of adenosine deaminase, 5''-nucleotidase, guanase, and cytidine deaminase enzymes in cancerous and non-cancerous human breast tissues." Breast Cancer Res. Treat. 37, 189-193.
Cho, D. S., Yang, W., Lee, J. T., Shiekhattar, R., Murray, J. M., and Nishikura, K. (2003). Requirement of Dimerization for RNA Editing Activity of Adenosine Deaminases Acting on RNA. J. Biol. Chem. 278, 17093-17102.
Christiansen, L. C., Schou, S. P., Nygaard and Saxild, H. H. (1997). "Xanthine metabolism in Bacillus subtilis: characterization of the xpt-pbuX operon and evidence for purine- and nitrogen-controlled expression of genes involved in xanthine salvage and catabolism." J. Bacteriol 179, 2540-2550.
Cruz-Ramos, H., Glaser, P. L., Wray, V. Jr. and Fisher, S. H. (1997). "The Bacillus subtilis ureABC operon." J. Bacteriol 179, 3371-3373.
Dietmann, S., Park, J. C., Notredame, Heger, A., Lappe, M., and Holm, L. (2001). "A fully automatic evolutionary classification of protein folds: Dali Domain Dictionary version 3." Nucleic Acids Res. 29, 55-57.
Durak, I., Beduk, Y., Kavutcu, M., Suzer, O., Yaman, O., Ozturk, H. S., Canbolat, O., and Ulutepe, S. (1997). "Activity of the enzymes participating in purine metabolism of cancerous and noncancerous human kidney tissues." Cancer Invest. 15, 212-216.
Fassbinder, F., Kist, M., and Bereswill, S. (2000). "Structural and functional analysis of the riboflavin synthesis genes encoding GTP cyclohydrolase II (ribA), DHBP synthase (ribBA), riboflavin synthase (ribC), and riboflavin deaminase/reductase (ribD) from Helicobacter pylori strain P1." FEMS Microbiol. Lett. 191, 191-197.
Fujiwara, S. and Noguchi, T. (1995). "Degradation of purines: only ureidoglycollate lyase out of four allantoin-degrading enzymes is present in mammals." Biochem. J. 312 , 315-318.
Hayashi, S., Jain, S., Chu, R., Alvares, K., Xu, B., Erfurth, F., Usuda, N.,
Rao, M. S., Reddy, S. K., Noguchi, T., and et al. (1994). "Amphibian allantoinase. Molecular cloning, tissue distribution, and functional expression." J. Biol. Chem. 269, 12269-12276.
Johansson, E., Mejlhede, N., Neuhard, J., and Larsen, S. (2002). "Crystal structure of the tetrameric cytidine deaminase from Bacillus subtilis at 2.0 Å resolution." Biochemistry 41, 2563-2570.
Keegan, L. P., Leroy, A., Sproul, D., and O''Connell, M. A. (2004). "Adenosine deaminases acting on RNA (ADARs): RNA-editing enzymes." Genome Biol. 5, 209-218.
Kievit, E., Bershad, E., Ng, E., Sethna, P., Dev, I., Lawrence, T. S., and Rehemtulla, A. (1999). "Superiority of yeast over bacterial cytosine deaminase for enzyme/prodrug gene therapy in colon cancer xenografts." Cancer Res. 59, 1417-1421.
Ko, T. P., Lin, J. J., Hu, C. Y., Hsu, Y. H., Wang A. H., and Liaw, S. H. (2003). "Crystal structure of yeast cytosine deaminase. Insights into enzyme mechanism and evolution." J. Biol. Chem. 278, 19111-19117.
Kumar, M. and Carmichael, G. G. (1997). "Nuclear antisense RNA induces extensive adenosine modifications and nuclear retention of target transcripts." Proc. Natl. Acad. Sci. U S A 94, 3542-3547.
Kuwahara, H., Araki, N., Makino, K., Masuko, N., Honda, S., Kaibuchi, K., Fukunaga, K., Miyamoto, E., Ogawa, M., and Saya, H. (1999). "A novel NE-dlg/SAP102-associated protein, p51-nedasin, related to the amidohydrolase superfamily, interferes with the association between NE-dlg/SAP102 and N-methyl-D-aspartate receptor." J. Biol. Chem. 274, 32204-32214.
Lewis, A. S. and Glantz, M. D. (1974). "Rabbit liver guanine deaminase. Chemical, physical, and kinetic properties." J. Biol. Chem. 249, 3862-3866.
Liou, J. Y., Krishnan, P., Hsieh, C. C., Dutschman, G. E., and Cheng, Y. C. (2003). "Assessment of the effect of phosphorylated metabolites of anti-human immunodeficiency virus and anti-hepatitis B virus pyrimidine analogs on the behavior of human deoxycytidylate deaminase." Mol. Pharmacol. 63, 105-110.
Liu, Y. and Eisenberg, D. (2002). "3D domain swapping: as domains continue to swap." Protein Sci. 11, 1285-1299.
Matsumoto, H., Ohta, S., Kobayashi, R., and Terawaki, Y. (1978).
"Chromosomal location of genes participating in the degradation of purines in Pseudomonas aeruginosa." Mol. Gen. Genet. 167, 165-176.
Matsunaga, H., Honda, H., Kubo, K., Sannomiya, K., Cui, X., Toyota, Y., Mori, T., Muguruma, N., Okahisa, T., Okamura, S., Shimizu, I., and Ito, S. (2003). "Clinical value of the determination of serum guanase activity in patients with chronic hepatitis type C." J. Med. Invest. 50, 64-71.
Muramatsu, M., Sankaranand, V. S., Anant, S., Sugai, M., Kinoshita, K., Davidson, N. O., Honjo, T. (1999) "Specific expression of activation induced cytidine deaminase (AID), a novel member of the RNA-editing deaminase family in germinal center B cells. " J. Biol. Chem. 274, 18470–18476.
Nygaard, P., Bested, S. M., Andersen, K. A. and Saxild, H. H. (2000). "Bacillus subtilis guanine deaminase is encoded by the yknA gene and is induced during growth with purines as the nitrogen source." Microbiology 146 , 3061-3069.
Porter, D. J. (2000). "Escherichia coli cytosine deaminase: the kinetics and thermodynamics for binding of cytosine to the apoenzyme and the Zn2+ holoenzyme are similar." Biochim. Biophys. Acta 1476, 239-252.
Richter, G., Fischer, M., Krieger, C., Eberhardt, S., Luttgen, H., Gerstenschlager, I., and Bacher, A. (1997). Biosynthesis of riboflavin: characterization of the bifunctional deaminase-reductase of Escherichia coli and Bacillus subtilis. J. Bacteriol. 179, 2022-2028.
Schaub, M., and Keller, W. (2002). RNA editing by adenosine deaminases generates RNA and protein diversity. Biochimie. 84, 791-803.
Wedekind, J. E., Dance, G. S., Sowden, M. P., and Smith, H. C. (2003). "Messenger RNA editing in mammals: new members of the APOBEC family seeking roles in the family business." Trends Genet. 19, 207-216.
Weiner, K. X., Weiner, R. S., Maley, F., and Maley, G. F. (1993). "Primary structure of human deoxycytidylate deaminase and overexpression of its functional protein in Escherichia coli." J. Biol. Chem. 268, 12983-12989.
Wolan, D. W., Greasley, S. E., Beardsley, G. P., and Wilson, I. A. (2002). "Structural insights into the avian AICAR transformylase mechanism." Biochemistry 41, 15505-15513.
Wu, X. W., Lee, C. C., Muzny, D. M., and Caskey, C. T. (1989). "Urate oxidase: primary structure and evolutionary implications." Proc. Natl. Acad. Sci. U S A 86, 9412-9416.
Xiang, S., Short, S. A., Wolfenden, R., and Carter, C. W. Jr. (1997). "The structure of the cytidine deaminase-product complex provides evidence for efficient proton transfer and ground-state destabilization." Biochemistry 36, 4768-4774.
Yuan, G., Bin, J. C., McKay, D. J., and Snyder, F. F. (1999). "Cloning and characterization of human guanine deaminase. Purification and partial amino acid sequence of the mouse protein." J. Biol. Chem. 274, 8175-8180.
許憶馨,2003,The crystal structure of yeast cytosine deaminase and its application in enzyme-prodrug gene therapy,國立陽明大學生物藥學研究所,碩士論文。
賴政宗,2004,Structural analysis of the cytidine deaminase superfamily: conservation, divergence and prediction,國立陽明大學遺傳學研究所,碩士論文。
第一頁 上一頁 下一頁 最後一頁 top