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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 詹迺立(Nei-Li Chan) | |
dc.contributor.author | Hsuan-Jen Liao | en |
dc.contributor.author | 廖宣任 | zh_TW |
dc.date.accessioned | 2021-06-17T07:40:22Z | - |
dc.date.available | 2020-03-05 | |
dc.date.copyright | 2019-03-05 | |
dc.date.issued | 2019 | |
dc.date.submitted | 2019-02-15 | |
dc.identifier.citation | 1 Cushnie, T. P., Cushnie, B. & Lamb, A. J. Alkaloids: an overview of their antibacterial, antibiotic-enhancing and antivirulence activities. Int J Antimicrob Agents 44, 377-386, doi:10.1016/j.ijantimicag.2014.06.001 (2014).
2 Dembitsky, V. M. Astonishing diversity of natural surfactants: 6. Biologically active marine and terrestrial alkaloid glycosides. Lipids 40, 1081-1105 (2005). 3 Ahmed, A. & Daneshtalab, M. Nonclassical biological activities of quinolone derivatives. J Pharm Pharm Sci 15, 52-72 (2012). 4 Kusano, M. et al. Nematicidal alkaloids and related compounds produced by the fungus Penicillium cf. simplicissimum. Biosci Biotechnol Biochem 64, 2559-2568, doi:10.1271/bbb.64.2559 (2000). 5 Simonetti, S. O., Larghi, E. L. & Kaufman, T. S. The 3,4-dioxygenated 5-hydroxy-4-aryl-quinolin-2(1H)-one alkaloids. Results of 20 years of research, uncovering a new family of natural products. Nat Prod Rep 33, 1425-1446, doi:10.1039/c6np00064a (2016). 6 Scherlach, K. & Hertweck, C. Discovery of aspoquinolones A-D, prenylated quinoline-2-one alkaloids from Aspergillus nidulans, motivated by genome mining. Org Biomol Chem 4, 3517-3520, doi:10.1039/b607011f (2006). 7 Rapoport, H. W. S. a. H. Mechanism of the transformation of cyclopenin to viridicatin. Journal of the American Chemical Society 91, 7, doi:10.1021/ja01050a026 (1969). 8 Uchida, R., Imasato, R., Tomoda, H. & Omura, S. Yaequinolones, new insecticidal antibiotics produced by Penicillium sp. FKI-2140. II. Structural elucidation. J Antibiot (Tokyo) 59, 652-658, doi:10.1038/ja.2006.87 (2006). 9 An, C. Y. et al. 4-Phenyl-3,4-dihydroquinolone derivatives from Aspergillus nidulans MA-143, an endophytic fungus isolated from the mangrove plant Rhizophora stylosa. J Nat Prod 76, 1896-1901, doi:10.1021/np4004646 (2013). 10 Ciegler, A. & Hou, C. T. Isolation of viridicatin from Penicillium palitans. Arch Mikrobiol 73, 261-267 (1970). 11 Cunningham, K. G. & Freeman, G. G. The isolation and some chemical properties of viridicatin, a metabolic product of Penicillium viridicatum Westling. Biochem J 53, 328-332 (1953). 12 Walsh, C. T., Haynes, S. W., Ames, B. D., Gao, X. & Tang, Y. Short pathways to complexity generation: fungal peptidyl alkaloid multicyclic scaffolds from anthranilate building blocks. ACS Chem Biol 8, 1366-1382, doi:10.1021/cb4001684 (2013). 13 Aboutabl, E. A. & Luckner, M. Cyclopeptine dehydrogenase in Penicillium cyclopium. Phytochemistry 14, 2573-2577, (1975) https://doi.org/10.1016/0031-9422(75)85227-7. 14 Voigt, S. & Luckner, M. Dehydrocyclopeptine epoxidase from Penicillium cyclopium. Phytochemistry 16, 1651-1655, (1977) https://doi.org/10.1016/0031-9422(71)85063-X. 15 Luckner, M. [On the synthesis of quinoline alkaloids in plants. 2. Fermentativ conversion of the penicillin alkaloids cyclopenin and cyclopenol to viridicatin and viridicatol]. Eur J Biochem 2, 74-78 (1967). 16 Ishikawa, N. et al. Non-heme dioxygenase catalyzes atypical oxidations of 6,7-bicyclic systems to form the 6,6-quinolone core of viridicatin-type fungal alkaloids. Angew Chem Int Ed Engl 53, 12880-12884, doi:10.1002/anie.201407920 (2014). 17 Martinez, S. & Hausinger, R. P. Catalytic Mechanisms of Fe(II)- and 2-Oxoglutarate-dependent Oxygenases. J Biol Chem 290, 20702-20711, doi:10.1074/jbc.R115.648691 (2015). 18 Abu-Omar, M. M., Loaiza, A. & Hontzeas, N. Reaction mechanisms of mononuclear non-heme iron oxygenases. Chem Rev 105, 2227-2252, doi:10.1021/cr040653o (2005). 19 Costas, M., Mehn, M. P., Jensen, M. P. & Que, L., Jr. Dioxygen activation at mononuclear nonheme iron active sites: enzymes, models, and intermediates. Chem Rev 104, 939-986, doi:10.1021/cr020628n (2004). 20 Decker, A. & Solomon, E. I. Dioxygen activation by copper, heme and non-heme iron enzymes: comparison of electronic structures and reactivities. Curr Opin Chem Biol 9, 152-163, doi:10.1016/j.cbpa.2005.02.012 (2005). 21 Pau, M. Y., Lipscomb, J. D. & Solomon, E. I. Substrate activation for O2 reactions by oxidized metal centers in biology. Proc Natl Acad Sci U S A 104, 18355-18362, doi:10.1073/pnas.0704191104 (2007). 22 Joosten, V. & van Berkel, W. J. Flavoenzymes. Curr Opin Chem Biol 11, 195-202, doi:10.1016/j.cbpa.2007.01.010 (2007). 23 Hausinger, R. P. FeII/alpha-ketoglutarate-dependent hydroxylases and related enzymes. Crit Rev Biochem Mol Biol 39, 21-68, doi:10.1080/10409230490440541 (2004). 24 Loenarz, C. & Schofield, C. J. Expanding chemical biology of 2-oxoglutarate oxygenases. Nat Chem Biol 4, 152-156, doi:10.1038/nchembio0308-152 (2008). 25 Price, J. C., Barr, E. W., Tirupati, B., Bollinger, J. M., Jr. & Krebs, C. The first direct characterization of a high-valent iron intermediate in the reaction of an alpha-ketoglutarate-dependent dioxygenase: a high-spin FeIV complex in taurine/alpha-ketoglutarate dioxygenase (TauD) from Escherichia coli. Biochemistry 42, 7497-7508, doi:10.1021/bi030011f (2003). 26 Riggs-Gelasco, P. J. et al. EXAFS Spectroscopic Evidence for an FeO Unit in the Fe(IV) Intermediate Observed during Oxygen Activation by Taurine:α-Ketoglutarate Dioxygenase. Journal of the American Chemical Society 126, 8108-8109, doi:10.1021/ja048255q (2004). 27 Proshlyakov, D. A., Henshaw, T. F., Monterosso, G. R., Ryle, M. J. & Hausinger, R. P. Direct detection of oxygen intermediates in the non-heme Fe enzyme taurine/alpha-ketoglutarate dioxygenase. J Am Chem Soc 126, 1022-1023, doi:10.1021/ja039113j (2004). 28 Schofield, C. J. & Zhang, Z. Structural and mechanistic studies on 2-oxoglutarate-dependent oxygenases and related enzymes. Curr Opin Struct Biol 9, 722-731 (1999). 29 Gorres, K. L. & Raines, R. T. Prolyl 4-hydroxylase. Crit Rev Biochem Mol Biol 45, 106-124, doi:10.3109/10409231003627991 (2010). 30 Vaz, F. M. & Wanders, R. J. Carnitine biosynthesis in mammals. Biochem J 361, 417-429 (2002). 31 Wanders, R. J., Komen, J. & Ferdinandusse, S. Phytanic acid metabolism in health and disease. Biochim Biophys Acta 1811, 498-507, doi:10.1016/j.bbalip.2011.06.006 (2011). 32 Ozer, A. & Bruick, R. K. Non-heme dioxygenases: cellular sensors and regulators jelly rolled into one? Nat Chem Biol 3, 144-153, doi:10.1038/nchembio863 (2007). 33 Taabazuing, C. Y., Hangasky, J. A. & Knapp, M. J. Oxygen sensing strategies in mammals and bacteria. J Inorg Biochem 133, 63-72, doi:10.1016/j.jinorgbio.2013.12.010 (2014). 34 Shen, L., Song, C. X., He, C. & Zhang, Y. Mechanism and function of oxidative reversal of DNA and RNA methylation. Annu Rev Biochem 83, 585-614, doi:10.1146/annurev-biochem-060713-035513 (2014). 35 Trewick, S. C., Henshaw, T. F., Hausinger, R. P., Lindahl, T. & Sedgwick, B. Oxidative demethylation by Escherichia coli AlkB directly reverts DNA base damage. Nature 419, 174-178, doi:10.1038/nature00908 (2002). 36 Johansson, C. et al. The roles of Jumonji-type oxygenases in human disease. Epigenomics 6, 89-120, doi:10.2217/epi.13.79 (2014). 37 Mosammaparast, N. & Shi, Y. Reversal of histone methylation: biochemical and molecular mechanisms of histone demethylases. Annu Rev Biochem 79, 155-179, doi:10.1146/annurev.biochem.78.070907.103946 (2010). 38 Cheng, A. X., Han, X. J., Wu, Y. F. & Lou, H. X. The function and catalysis of 2-oxoglutarate-dependent oxygenases involved in plant flavonoid biosynthesis. Int J Mol Sci 15, 1080-1095, doi:10.3390/ijms15011080 (2014). 39 Hamed, R. B. et al. The enzymes of beta-lactam biosynthesis. Nat Prod Rep 30, 21-107, doi:10.1039/c2np20065a (2013). 40 Aik, W. S. C., Rashed & Clifton, I.J. & Hopkinson, R.J. & Leissing, T & McDonough, Michael & Nowak, R & Schofield, C.J. & Walport, Louise. . Introduction to structural studies on 2-oxoglutarate-dependent oxygenases and related enzymes., 15, doi:10.1039/9781782621959-00059. (2015). 41 Hegg, E. L. & Que, L., Jr. The 2-His-1-carboxylate facial triad--an emerging structural motif in mononuclear non-heme iron(II) enzymes. Eur J Biochem 250, 625-629 (1997). 42 Aik, W., McDonough, M. A., Thalhammer, A., Chowdhury, R. & Schofield, C. J. Role of the jelly-roll fold in substrate binding by 2-oxoglutarate oxygenases. Curr Opin Struct Biol 22, 691-700, doi:10.1016/j.sbi.2012.10.001 (2012). 43 Clifton, I. J. et al. Structural studies on 2-oxoglutarate oxygenases and related double-stranded beta-helix fold proteins. J Inorg Biochem 100, 644-669, doi:10.1016/j.jinorgbio.2006.01.024 (2006). 44 Brauer, A., Beck, P., Hintermann, L. & Groll, M. Structure of the Dioxygenase AsqJ: Mechanistic Insights into a One-Pot Multistep Quinolone Antibiotic Biosynthesis. Angew Chem Int Ed Engl 55, 422-426, doi:10.1002/anie.201507835 (2016). 45 Hanauske-Abel, H. M. & Gunzler, V. A stereochemical concept for the catalytic mechanism of prolylhydroxylase: applicability to classification and design of inhibitors. J Theor Biol 94, 421-455 (1982). 46 Abad, J. L., Camps, F. & Fabrias, G. Is Hydrogen Tunneling Involved in AcylCoA Desaturase Reactions? The Case of a Delta(9) Desaturase That Transforms (E)-11-Tetradecenoic Acid into (Z,E)-9,11-Tetradecadienoic Acid This work was supported by Comision Asesora de Investigacion Cientifica y Tecnica (grant AGF 98-0844), Comissionat per a Universitats i Recerca from the Generalitat de Catalunya (grant 97SGR-0021) and SEDQ S.A. We thank Prof. Nigel S. Scrutton (University of Leicester, UK) and Dr. Francisco J. Sanchez-Baeza (IIQAB, Barcelona, Spain) for helpful discussions, Dr. Josefina Casas and Dr. Antonio Delgado for critically reading the manuscript, and German Lazaro for rearing the insects used in this study. J.L.A. thanks the Spanish Ministry of Education and Science for a Postdoctoral Reincorporation Contract. Angew Chem Int Ed Engl 39, 3279-3281 (2000). 47 Meunier, B., de Visser, S. P. & Shaik, S. Mechanism of oxidation reactions catalyzed by cytochrome p450 enzymes. Chem Rev 104, 3947-3980, doi:10.1021/cr020443g (2004). 48 Solomon, E. I., Decker, A. & Lehnert, N. Non-heme iron enzymes: contrasts to heme catalysis. Proc Natl Acad Sci U S A 100, 3589-3594, doi:10.1073/pnas.0336792100 (2003). 49 Britsch, L. Purification and characterization of flavone synthase I, a 2-oxoglutarate-dependent desaturase. Arch Biochem Biophys 282, 152-160 (1990). 50 Cooper, H. L. et al. Parallel and competitive pathways for substrate desaturation, hydroxylation, and radical rearrangement by the non-heme diiron hydroxylase AlkB. J Am Chem Soc 134, 20365-20375, doi:10.1021/ja3059149 (2012). 51 Abad, J. L., Camps, F. & Fabrias, G. Substrate-dependent stereochemical course of the (Z)-13-desaturation catalyzed by the processionary moth multifunctional desaturase. J Am Chem Soc 129, 15007-15012, doi:10.1021/ja0751936 (2007). 52 Whittle, E. J., Tremblay, A. E., Buist, P. H. & Shanklin, J. Revealing the catalytic potential of an acyl-ACP desaturase: tandem selective oxidation of saturated fatty acids. Proc Natl Acad Sci U S A 105, 14738-14743, doi:10.1073/pnas.0805645105 (2008). 53 Reilly, C. A. et al. Metabolism of capsaicin by cytochrome P450 produces novel dehydrogenated metabolites and decreases cytotoxicity to lung and liver cells. Chem Res Toxicol 16, 336-349, doi:10.1021/tx025599q (2003). 54 Ji, L. et al. Drug metabolism by cytochrome p450 enzymes: what distinguishes the pathways leading to substrate hydroxylation over desaturation? Chemistry 21, 9083-9092, doi:10.1002/chem.201500329 (2015). 55 Newcomb, M. et al. Cytochrome P450-Catalyzed Hydroxylation of Mechanistic Probes that Distinguish between Radicals and Cations. Evidence for Cationic but Not for Radical Intermediates. Journal of the American Chemical Society 122, 2677-2686, doi:10.1021/ja994106+ (2000). 56 J.M., J., Bollinger, & Chang, W.-C & Matthews, M.L. & Martinie, R.J. & Boal, A.K. & Krebs, C. . Mechanisms of 2-oxoglutarate-dependent oxygenases: The hydroxylation paradigm and beyond., 27, doi:10.1039/9781782621959-00095 (2015). 57 Wang, C. et al. Evidence that the fosfomycin-producing epoxidase, HppE, is a non-heme-iron peroxidase. Science 342, 991-995, doi:10.1126/science.1240373 (2013). 58 Li, J., van Belkum, M. J. & Vederas, J. C. Functional characterization of recombinant hyoscyamine 6beta-hydroxylase from Atropa belladonna. Bioorg Med Chem 20, 4356-4363, doi:10.1016/j.bmc.2012.05.042 (2012). 59 Hollenhorst, M. A. et al. The nonribosomal peptide synthetase enzyme DdaD tethers N(beta)-fumaramoyl-l-2,3-diaminopropionate for Fe(II)/alpha-ketoglutarate-dependent epoxidation by DdaC during dapdiamide antibiotic biosynthesis. J Am Chem Soc 132, 15773-15781, doi:10.1021/ja1072367 (2010). 60 Seo, M. J., Zhu, D., Endo, S., Ikeda, H. & Cane, D. E. Genome mining in Streptomyces. Elucidation of the role of Baeyer-Villiger monooxygenases and non-heme iron-dependent dehydrogenase/oxygenases in the final steps of the biosynthesis of pentalenolactone and neopentalenolactone. Biochemistry 50, 1739-1754, doi:10.1021/bi1019786 (2011). 61 Chang, W. C., Li, J., Lee, J. L., Cronican, A. A. & Guo, Y. Mechanistic Investigation of a Non-Heme Iron Enzyme Catalyzed Epoxidation in (-)-4'-Methoxycyclopenin Biosynthesis. J Am Chem Soc 138, 10390-10393, doi:10.1021/jacs.6b05400 (2016). 62 Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol 276, 307-326 (1997). 63 McCoy, A. J. et al. Phaser crystallographic software. J Appl Crystallogr 40, 658-674, doi:10.1107/S0021889807021206 (2007). 64 Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr 67, 235-242, doi:10.1107/S0907444910045749 (2011). 65 Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr D Biol Crystallogr 68, 352-367, doi:10.1107/S0907444912001308 (2012). 66 Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66, 486-501, doi:10.1107/S0907444910007493 (2010). 67 Evans, G. & Pettifer, R. F. CHOOCH: a program for deriving anomalous-scattering factors from X-ray fluorescence spectra. Journal of Applied Crystallography 34, 82-86, doi:Doi 10.1107/S0021889800014655 (2001). 68 Liao, H. J. et al. Insights into the Desaturation of Cyclopeptin and its C3 Epimer Catalyzed by a non-Heme Iron Enzyme: Structural Characterization and Mechanism Elucidation. Angew Chem Int Ed Engl 57, 1831-1835, doi:10.1002/anie.201710567 (2018). 69 Dey, A. et al. X-ray absorption spectroscopy and density functional theory studies of [(H3buea)FeIII-X]n- (X = S2-, O2-, OH-): comparison of bonding and hydrogen bonding in oxo and sulfido complexes. J Am Chem Soc 128, 9825-9833, doi:10.1021/ja061618x (2006). 70 MacBeth, C. E. et al. Utilization of hydrogen bonds to stabilize M-O(H) units: synthesis and properties of monomeric iron and manganese complexes with terminal oxo and hydroxo ligands. J Am Chem Soc 126, 2556-2567, doi:10.1021/ja0305151 (2004). 71 Smith, J. M. et al. N-O bond homolysis of an iron(II) TEMPO complex yields an iron(III) oxo intermediate. J Am Chem Soc 134, 6516-6519, doi:10.1021/ja211882e (2012). 72 Song, X., Lu, J. & Lai, W. Mechanistic insights into dioxygen activation, oxygen atom exchange and substrate epoxidation by AsqJ dioxygenase from quantum mechanical/molecular mechanical calculations. Phys Chem Chem Phys 19, 20188-20197, doi:10.1039/c7cp02687k (2017). 73 Mitchell, A. J. et al. Visualizing the Reaction Cycle in an Iron(II)- and 2-(Oxo)-glutarate-Dependent Hydroxylase. J Am Chem Soc 139, 13830-13836, doi:10.1021/jacs.7b07374 (2017). 74 Hoffart, L. M., Barr, E. W., Guyer, R. B., Bollinger, J. M., Jr. & Krebs, C. Direct spectroscopic detection of a C-H-cleaving high-spin Fe(IV) complex in a prolyl-4-hydroxylase. Proc Natl Acad Sci U S A 103, 14738-14743, doi:10.1073/pnas.0604005103 (2006). 75 Galonic, D. P., Barr, E. W., Walsh, C. T., Bollinger, J. M., Jr. & Krebs, C. Two interconverting Fe(IV) intermediates in aliphatic chlorination by the halogenase CytC3. Nat Chem Biol 3, 113-116, doi:10.1038/nchembio856 (2007). 76 Fujimori, D. G. et al. Spectroscopic evidence for a high-spin Br-Fe(IV)-oxo intermediate in the alpha-ketoglutarate-dependent halogenase CytC3 from Streptomyces. J Am Chem Soc 129, 13408-13409, doi:10.1021/ja076454e (2007). 77 Matthews, M. L. et al. Substrate-triggered formation and remarkable stability of the C-H bond-cleaving chloroferryl intermediate in the aliphatic halogenase, SyrB2. Biochemistry 48, 4331-4343, doi:10.1021/bi900109z (2009). 78 Chang, W. C. et al. Mechanism of the C5 stereoinversion reaction in the biosynthesis of carbapenem antibiotics. Science 343, 1140-1144, doi:10.1126/science.1248000 (2014). 79 Borowski, T., Bassan, A. & Siegbahn, P. E. Mechanism of dioxygen activation in 2-oxoglutarate-dependent enzymes: a hybrid DFT study. Chemistry 10, 1031-1041, doi:10.1002/chem.200305306 (2004). 80 Topol, I. A. et al. Quantum chemical modeling of reaction mechanism for 2-oxoglutarate dependent enzymes: effect of substitution of iron by nickel and cobalt. J Phys Chem A 110, 4223-4228, doi:10.1021/jp055633k (2006). 81 Wojcik, A., Radon, M. & Borowski, T. Mechanism of O2 Activation by alpha-Ketoglutarate Dependent Oxygenases Revisited. A Quantum Chemical Study. J Phys Chem A 120, 1261-1274, doi:10.1021/acs.jpca.5b12311 (2016). | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/73532 | - |
dc.description.abstract | 小巢狀麴菌之AsqJ為一非血基質鐵/-酮戊二酸依賴型雙氧化酶,負責催化受質cyclopeptin連續性的脫氫以及環氧化反應以形成產物cyclopenin。 此氧化反應起始於氧分子與二價鐵離子的結合,再藉由氧分子誘發-酮戊二酸之氧化脫羧反應、使其裂解為二氧化碳以及琥珀酸,同時形成具高氧化力之四價鐵與單氧鍵結的重要中間產物(ferryl-oxo intermediate),進而輔助受質之脫氫或環氧化。目前已解出的AsqJ 晶體結構包括以鎳取代活性中心鐵的原態構形、及其與受質cyclopeptin或受質衍生物形成的複合體。然而,對於AsqJ所催化之反應的詳細機制則仍待進一步闡明。在本篇研究中,我們首先解出了AsqJ分別與受質cyclopeptin及其C3-表異構物(即D-cyclopeptin)結合的兩個高解析度複合體結構。X-光近緣吸收光譜顯示與受質cyclopeptin結合的AsqJ複合體當中有鐵的訊號。這意味著此AsqJ結構確為與鐵結合的活性態構形。比較此二結構中cyclopeptin與D-cyclopeptin的構形可知,cyclopeptin之三號碳的氫原子以及十號碳上其中一個氫原子皆朝向鐵原子中心; 然而D-cyclopeptin只有十號碳上其中一個氫原子朝向鐵原子中心,而三號碳上的氫原子是遠離鐵原子中心的。基於此結果可推論AsqJ所催化的脫氫反應是透過四價鐵與單氧鍵結的過渡狀態先在十號碳的位置進行氫原子轉移,後續的反應會經形成碳正離子或是羥基化過渡狀態完成。其次,我們分別利用結晶後浸換或同時進行晶體內反應以及共結晶的實驗方法,得到多個AsqJ在催化環氧化反應過程中可能出現的反應中間物結構、以及兩種與AsqJ結合時呈現不同構型的2-CF3受質類似物結構。結構分析指出AsqJ所催化的環氧化反應可能是經由形成三價鐵與單氧鍵結的過渡狀態,再由此單氧原子與受質上的十號碳形成單鍵鍵結,最後形成環氧產物。有趣的是,結構疊合分析顯示構型一的2-CF3上的十號碳與單氧原子之間的距離(2.6 Å)較在形成O-C10鍵結(2.0 Å)的反應中間物遠。此結果與2-CF3受質類似物較不易進行環氧化反應的結果一致。我們也解出兩個有氧分子結合且分別與受質cyclopeptin以及dehydrocyclopeptin結合的AsqJ複合體結構、反應中間物 bicyclic Fe(Ⅳ)-peroxyhemiketal intermediate結構、以硫酸氧釩進行鐵取代所模擬的四價鐵與單氧鍵結過渡狀態結構、以及藉由琥珀酸進行置換所獲得模擬單氧原子重組後的四價鐵與單氧鍵結過度狀態的結構。這些實驗結果對於-酮戊二酸依賴型雙氧化酶進行雙氧活化的機制提供更詳細的資訊。總結以上的研究成果,這些先前尚未被發現的晶體結構完整地描繪出AsqJ所催化進行脫氫與環氧化反應的機制。 | zh_TW |
dc.description.abstract | AsqJ is a non-heme FeII/2-oxoglutarate-dependent dioxygenase recently discovered in Aspergillus nidulans, which catalyzes the sequential desaturation and epoxidation on the substrate cyclopeptin to produce cyclopenin. Each AsqJ-mediated oxidation step is accompanied by the breakdown of 2-oxoglutarate (2OG) into succinate and CO2, which results in the formation of a high-valent FeⅣ-oxo species as a key intermediate. Crystal structures of the AsqJ in its NiII-substituted state and in complexes with cyclopeptin and its analogs have already been determined. However, a more complete and detailed mechanistic picture of the AsqJ-catalyzed reactions has remained to be further explored. Here, we report the high-resolution crystal structures of iron-bound AsqJ with cyclopeptin and its C3-epimer (D-cyclopeptin), respectively. An XANES spectrum collected from an AsqJ•Fe•2OG•cyclopeptin crystal unambiguously shows the presence of iron in the crystal. This structure provides the first visualization of AsqJ in its native, Fe-bound form. More importantly, different from the binding configuration of cyclopeptin where the C3-H and one of the C10-Hs are poised toward the iron center, in the structure of AsqJ•Fe•2OG•D-cyclopeptin, the C3-H points away from the iron center. These findings suggest that a pathway involving hydrogen atom abstraction at the C10 position of the substrate by a short-lived Fe(IV)-oxo species and the subsequent formation of a carbocation or a hydroxylated intermediate more likely accounts for AsqJ-catalyzed desaturation. We have also determined several structures on AsqJ that may correspond to intermediate states occurred during the epoxidation step, two different binding conformations of 2-CF3 (an analog of cyclopeptin) in complex with AsqJ, and structures resulted from in-crystal reaction. The two intermediate structures of AsqJ and a product-bound AsqJ complex suggest that the AsqJ-catalyzed epoxidation may go through ferric-oxo formation followed by O-C10 bond formation and epoxide production. Interestingly, superimposition analysis shows that the distance from the oxo group to C10 of substrate in conformation 1 of bound 2-CF3 analog (2.6 Å) is longer than the 2.0 Å seen in the intermediate showing O-C10 bond formation. This result may explain why 2-CF3 is less reactive toward AsqJ-catalyzed epoxidation. Finally, crystal structures of two O2 –bound AsqJ complexes with cyclopeptin and dehydrocyclopeptin, the bicyclic Fe(Ⅳ)-peroxyhemiketal intermediate, the Fe(Ⅳ)-oxo intermediate mimicked by VOSO4 replacement, and the Fe(Ⅳ)-oxo intermediate via rearrangement mimicked by soaking succinate are determined. Together, these results not only depict the mechanism of AsqJ-catalyzed desaturation and epoxidation, but also provide more detailed information on dioxygen activation of 2OG-dependent dioxygenase superfamily. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T07:40:22Z (GMT). No. of bitstreams: 1 ntu-108-D03442002-1.pdf: 6669291 bytes, checksum: a4d74334fb6387af1d0855879596c2a3 (MD5) Previous issue date: 2019 | en |
dc.description.tableofcontents | 口試委員會審定書 I
誌謝 II 中文摘要 III Abstract V Contents VIII List of Figures XI List of Tables XIII 1. Introduction 1 1.1. AsqJ dioxygenase: an essential enzyme for the biosynthesis of viridicatin-type quinolone alkaloids 2 1.2. Non-heme iron (Fe2+)/α-ketoglutarate-dependent dioxygenases 5 1.3. The consensus mechanism of hydroxylation in non-heme Fe(II)/2OG dependent dioxygenases 8 1.4. Functions of the AsqJ dioxygenase and a proposed mechanism of AsqJ-catalyzed sequential oxidative reactions 10 1.5. Specific aims of this study 12 2. Methods and Materials 15 2.1. Plasmid for expression of recombinant AsqJ 16 2.2. Expression and purification of AsqJ 16 2.3. Protein crystallization 17 2.4. Post-crystallization soaking 19 2.5. Post-crystallization soaking along with ascorbate treatment 20 2.6. Structure determination 20 2.7. X-ray absorption near-edge structure (XANES) spectroscopy 22 3. Results and Discussion 23 3.1. Structure determination of AsqJ·Fe·2OG·cyclopeptin and AsqJ·Fe·2OG·D-cyclopeptin quaternary complex 24 3.2. Structural characterization of AsqJ-catalyzed desaturation 26 3.3. Structure determination of AsqJ·Fe·2OG·dehydrocyclopeptin·O2 quinary complex………………………………………………………………………….....28 3.4. Mechanistic depiction of AsqJ-catalyzed epoxidation: structural characterizations of AsqJ·Fe-O·succinate·dehydrocyclopeptin, AsqJ·Fe·succinate·dehydrocyclopeptin (O-C10 bond formation) and AsqJ·Fe·2OG·cyclopepnin complexe 29 3.5. AsqJ-mediated epoxidation reactivity is susceptible to electron withdrawing ability on phenyl ring of olefin substarte: structure determination of AsqJ·Fe·tartrate·2-CF3 quaternary complex…………...........……………………..32 3.6. Mechanistic insights into the AsqJ-catalyzed dioxygen activation: structure determination of AsqJ·Fe·2OG·cyclopeptin·O2, AsqJ·Fe·peoxyhemiketal·cyclopeptin, AsqJ·VO·succinate·cyclopeptin, AsqJ·Fe·succinate·cyclopeptin and AsqJ·Fe-O·succinate·dehydrocyclopeptin complexes 34 4. Conclusion 40 5. Figures 43 6. Tables 76 7. References 82 8. Publication 90 | |
dc.language.iso | zh-TW | |
dc.title | 以結構觀點深入探討AsqJ所催化連續性的氧化反應機制 | zh_TW |
dc.title | Structural insight into the mechanism of AsqJ-catalyzed consecutive oxidation reactions | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-1 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 蕭傳鐙,李宗璘,簡敦誠,徐駿森,曾秀如 | |
dc.subject.keyword | 非血基質鐵/α-酮戊二酸依賴型雙氧化?,雙氧活化,脫氫反應,環氧化反應, | zh_TW |
dc.subject.keyword | non-heme FeII/2-oxoglutarate-dependent dioxygenase,dioxygen activation,desaturation,epoxidation, | en |
dc.relation.page | 95 | |
dc.identifier.doi | 10.6342/NTU201900608 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2019-02-15 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
顯示於系所單位: | 生物化學暨分子生物學科研究所 |
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