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研究生：	江志祥 Chiang, Chih-Hsiang
論文名稱：	利用細胞色素P450 BM3調控含氟化合物進行氧化時位置選擇性與其酵素動力學探討之研究 Regio-Selective Hydrolation of C12-C15 Fatty Acid with Fluorinated Substituents by Cytochrome P450 BM3
指導教授：	黃福永 Huang, Fu-Yung 俞聖法 Yu, Sheng-Fa
學位類別：	博士 Doctor
系所名稱：	理學院 - 化學系 Department of Chemistry
論文出版年：	2013
畢業學年度：	102
語文別：	英文
論文頁數：	89
中文關鍵詞：	細胞色素P450 BM3 、氟、位置選擇性、酵素動力學
外文關鍵詞：	Cytochrome P450, Regio-Selective, Fluorinated, Michaelis-Menten Kinetic
相關次數：	點閱：159 下載：2
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一般而言對於C-H 鍵的氧化是需要在高溫高壓下才可以進行的反應(烷類鍵的解
離能是98 kcal/mol )，所以要以傳統的有機化學的方式進行此反應是十分不容易；然而在生物界的系統當中許多的細菌可以在常溫常壓下進行羥基化反應 (hydroxylation reaction)，其中以又以我們所研究的菌種Bacillus megaterium 最為被廣泛的研究。在細胞色素P450 BM3(Cytochrome P450 BM3)中可以觀察到它對於長碳鏈脂肪酸(C12-C20)是具有高度的位置選擇性(會氧化在w-1,w-2 及w-3 位置上)與立體專一性(以R-form 二級醇為主要產物)。為了更加了解此生物系統的反應機制，我們除了用野生型(Wild-type)之外，也利用基因工程的方式突變三個重要的氨基酸位置(A74G F87V L188Q)，將其轉殖到大腸桿菌中加以大量表現與培養放大後純化出所需重要之蛋白質酵素，並使用長碳鏈含氟之脂肪酸當成受質去進行酵素反應，利用氣相層析儀與質譜儀的技術去鑑定出羥基化反應後的各種產物；我們也嘗試利用動力學實驗的方式去觀察各受質的催化效果，其研究結果發現，依數據來看當受質接上氟原子之後會使得耦合效率(coupling efficiency)增加、Michaelis-Menten constant(Km)降低、turnover frequencies (kcat) 提高及活化能(G≠)降低等效應，此外，當脂肪酸接氟原子之後，除了原本與Arg47/Tyr51 作用力最強的氫鍵仍會將脂肪酸的酸基固定住之外，包含紫質活性中心的酵素空間(M zone)則與末端的氟原子取代形成特定的凡德瓦作用力(van der Waals interactions)，進而驅使更佳的位置選擇性；其中，末端甲基經三個氟原子取代後的月桂酸，可達到羥基化近100%發生在w-3 的位置。藉由此研究的啟發，在未來希望可以利用這樣簡單又有效率的方式，合成出更多有機化合物，進而達到綠色化學與藥物化學上的應用。

In general, oxidation of the C-H bond occurs at high temperature and pressure (alkane bond dissociation energy is 98 kcal/mol) and hardly can be achieved through conventional organic synthesis. However, in many biological systems, this reaction can be carried out under atmospheric pressure at room temperature. Among these systems, cytochrome P450 BM3 from Bacillus megaterium stands out for its highly regio- and stereo-selective hydroxylation of C-H bonds at w-1,w-2 or w-3 of long chain fatty acids (C12-C20) with R-form alcohols as the main products. To understand the driving forces for its reaction the wild-type cytochrome P450 BM3 and another mutant (A74G F87V L188Q were overexpressed in E. coli and purified. Long-chain fatty acids and designed fluorinated counterparts were used to examine the effect from fluorine substituents. Products formation is verified by gas chromatography and mass spectrometry to compare the regio-/stereo- selectivity; spin-shift and kinetics experiments were also performed to analyze the catalytic process; NADPH consumption rate and product formation rate. Results here shows that, despite its minute effect on the binding affinity resulted from fluorine substituents, the higher turnover frequency and the lower activation energy (G≠)indicate that the unique van der Waals interactions indeed play important roles in the control of regio-/stereo-selective hydroxylation on fatty acids by P450 BM3. Based on these findings, the controlled hydroxylation by P450 BM3 can be fine-tuned in the future in hopes to develop simple and efficient ways to synthesize higher value organic compounds, thus achieving green chemistry and pharmaceutical applications.

中文摘要………………………………………1
Abstract………………………………………2
誌謝………………………………………3
Contents………………………………………5
Legends of the Figures………………………………………8
Legends of the Tables………………………………………13
Legends of the Schemes………………………………………14
Chapter 1 : Introduction………………………………………15
1.1	Classes of the Cytochrome P450 Superfamily…………15
1.2	The Catalytic Cycle of Cytochrome P450 BM3…………16
1.3	The Hydroxylation Mechanism of Cytochrome P450……17
1.4	The Crystal Structure of Cytochrome P450 BM3………18
1.5	The Functional Roles of the Key Residues in Cytochrome P450 BM3………………………………………19
1.5.1	Arg47 and Tyr51……………………………………………………19
1.5.2	Phe87……………………………………………………………………21
1.5.3	Thr268、Glu267 and Ala264………………………………………21
1.6	The Structural Insights Implicated for the Regio- and Stereo-selectivity of Substrates in Cytochrome P450 BM3………………………………………22
1.7	The Effects of Fluorinated Substituents (Substrates) in Biological System………………………………………25
1.8	The Specific Aims…………………………………………………26
Chapter 2 : Synthesis of Fluorinated Fatty Acid………………28
2.1 Background……………………………………………………………………28
2.2 Mechanism of Fluorination……………………………………………28
2.3 Preparation of Fluorinated Alkanes and Fatty Acids……29
2.4 Experimental Details for the Preparation of the Fluorinated Fatty Acids and Alkanes…………………………………30
2.4.1 Preparation of 1-Fluoro and 1,12-Difluorododecanes (9 and 10)………………………………………30
2.4.2 Preparation of 12-Fluorododecanoic Acid (5) and 12,12-
Difluorododecanoic Acid (6)………………………………………………32
2.4.3 Preparation of 12,12,12-Trifluorododecanoic Acid (7) and 15,15,15-
Trifluoropentadecanoic Acid (8)…………………………………………35
Chapter 3: Purification of Cytochrome P450 BM3…………………39
3.1 Methods for DNA Manipulation and Mutagenesis Studies, Recombinant Protein Expression and Purification of P450 BM3…………………………………………39
3.2 Materials……………………………………………………………………40
3.3 Instrumentations…………………………………………………………43
3.4 Preparation of Bio-related Materials…………………………44
3.4.1 Purification of Wild Type P450 BM3 and its variant 3mt………………………………………44
3.4.2 The Quantification of Recombinant Cytochrome P450 BM3………………………………………46
3.4.3 Determination of NADPH Consumption Rate…………………47
3.4.4 Determination of Product Formation Rate…………………47
3.4.5 Determination of Product Distributions……………………48
Chapter 4: Enzymatic Conversion of Fluorinated Substrates………………………………………55
4.1 Enzymatic Conversion of the Fluorinated Substrates Mediated by Cytochrome P450 BM3 and 3mt……………………………55
4.2 Determination of Specific Activities for Substrates 1-10 from the Rates of NADPH Consumption and Product Formation………………………………………60
4.3 Determination of Product Distributions…………………………………………62
4.4 Data for (w-x) Hydroxylated Products with Trimethylsilyl Derivatization after Conversion of Fatty Acids 5–8 by Wild-Type P450 BM3………………………64
Chapter 5: Spin-Shift and Michaelis-Menten Kinetic Study…66
5.1 Background……………………………………………………………………66
5.2 Determination of Association Constants (KHS), High Spin Conversion (%HS) and Michaelis–Menten Kinetics………………66
5.3 Spin-Shift Studies……………………………………………………67
5.4 The Effects of Fluorinated Substrates on the Kinetic Parameters……………………………………………………………………73
Chapter 6: Discussion…………………………………………………………79
Chapter 7: Conclusion…………………………………………………………83
Rferences……………………………………………85
                                    

(1)Roberts, G. A.; Grogan, G.; Greter, A.; Flitsch, S. L.; Turner, N. J. J. Bacteriol. 2002, 184, 3898.
(2)Haines, D. C.; Sevrioukova, I. F.; Peterson, J. A. Biochemistry 2000, 39, 9419.
(3)Noble, M. A.; Miles, C. S.; Chapman, S. K.; Lysek, D. A.; Mackay, A. C.; Reid, G. A.; Hanzlik, R. P.; Munro, A. W. Biochemistry J. 1999, 339, 371.
(4)Narhi, L. O.; Fulco, A. J. J. Biol. Chem. 1987, 262, 6689.
(5)Fasan, R.; Meharenna, Y. T.; Snow, C. D.; Poulos, T. L.; Arnold, F. H.
J. Mol. Biol. 2008, 383, 1069.
(6)Urlacher, V. B.; Schmid, R. D. Methods Enzymol 2004, 388, 208.
(7)Daff, S. N.; Chapman, S. K.; L.Turner, K.; Holt, R. A.; Govindaraj, S.; Poulos, T. L.; Munro, A. W. Biochemistry 1997, 36, 13816.
(8)Whitehouse, C. J.; Bell, S. G.; Wong, L. L. Chem. Soc. Rev. 2002, 41, 1218.
(9)Kitazume, T.; Haines, D. C.; Estabrook, R. W.; Chen, B.; Peterson, J. A. Biochemistry 2007, 46, 11892.
(10)Joyce, M. G.; Ekanem, I. S.; Roitel, O.; Dunford, A. J.; Neeli, R.; Girvan, H. M.; Baker, G. J.; Curtis, R. A.; Munro, A. W.; Leys, D. FEBS J. 2012, 279, 1694.
(11)Haines, D. C.; Tomchick, D. R.; Machius, M.; Peterson, J. A. Biochemistry 2001, 40, 13456.
(12)Aharoni, A.; Gaidukov, L.; Khersonsky, O.; Mc, Q. G. S.; Roodveldt, C.; Tawfik, D. S. Nature genetics 2005, 37, 73.
(13)Roiban, G.-D.; Reetz, M. T. Angew. Chem. Int. Ed. 2013, 52, 5439.
(14)Lewis, J. C.; Bastian, S.; Bennett, C. S.; Fu, Y.; Mitsuda, Y.; Chen, M. M.; Greenberg, W. A.; Wong, C. H.; Arnold, F. H. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 16550.
(15)Rentmeister, A.; Arnold, F. H.; Fasan, R. Nature chemical biology 2009, 5, 26.
(16)Lewis, D. F. V.; Jacobs, M. N.; Dickins, M. Drug Discovery Today 2004, 9, 530.
(17)Fishelovitch, D.; Shaik, S.; Wolfson, H. J.; Nussinov, R. J. Phys. Chem. B 2010, 114, 5964.
(18)Shaik, S.; Kumar, D.; Visser, S. l. P. d.; Altun, A.; Thiel, W. Chem. Rev. 2005, 105, 2279.
(19)Montellano, P. R. O. d. Chem. Rev. 2010, 110, 932.
(20)Shaik, S.; Cohen, S.; Wang, Y.; Chen, H.; Kumar, D.; Thiel, W. Chem. Rev. 2010, 110, 949.
(21)Feenstra, K. A.; Starikov, E. B.; Urlacher, V. B.; Commandeur, J. N.; Vermeulen, N. P. Protein sci. 2007, 16, 420.
(22)Jung, C. Biochim. Biophys. Acta 2011, 1814, 46.
(23)Sono, M.; Roach, M. P.; Coulter, E. D.; Dawson, J. H. Chem. Rev. 1996, 96, 2841.
(24)Meunier, B.; Visser, S. l. P. d.; Shaik, S. Chem. Rev. 2004, 104, 3947.
(25)Groves, J. T.; K.Shalyaev; Lee, J.; Kadish, K. M.; Smith, K. M.; Guilard, R. in The Porphyrin Handbook; Biochemistry and Binding: Activation of Small Molecules; Academic Press: New York, 2000.
(26)Lippard, S. J.; Berg, J. M. Principles of Bioinorganic chemistry 1994.
(27)Shilov, A. E.; Shul'pin, G. B. Chem. Rev. 1997, 97, 2879.
(28)Li, H. Y.; Poulos, T. L. Nat. Struct. Biol. 1997, 4, 140.
(29)Sevrioukova, I. F.; Li, H. Y.; Zhang, H.; Peterson, J. A.; Poulos, T. L. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 1863.
(30)Ravichandran, K. G.; Boddupalli, S. S.; Hasemann, C. A.; Peterson, J. A.; Deisenhofer, J. Science 1993, 261, 731.
(31)Haines, D. C.; Tomchick, D. R.; Machius, M.; Peterson, J. A. Biochemistry 2001, 40, 13456.
(32)Ost, T. W. B.; Miles, C. S.; Murdoch, J.; Cheung, Y.-F.; Reid, G. A.; Chapman, S. K.; Munro, A. W. FEBS J.2000, 486, 173.
(33)Li., H.; Poulos, T. L. Nat. struct. mol. biol. 1997, 4, 140.
(34)Li, H.; Poulos, T. Biochimine 1996, 78, 695.
(35)Hegde, A.; Haines, D. C.; Bondlela, M.; Chen, B.; Schaffer, N.; Tomchick, D. R.; Machius, M.; Nguyen, H.; Chowdhary, P. K.; Stewart, L.; Lopez, C.; Peterson, J. A. Biochemistry 2007, 46, 14010.
(36)Haines, D. C.; Hegde, A.; Chen, B.; Zhao, W.; Bondlela, M.; Humphreys, J. M.; Mullin, D. A.; Tomchick, D. R.; Machius, M.; Peterson, J. A. Biochemistry 2011, 50, 8333.
(37)Wu, L. L.; Yang, C. L.; Lo, F. C.; Chiang, C. H.; Chang, C. W.; Ng, K. Y.; Chou, H. H.; Hung, H. Y.; Chan, S. I.; Yu, S. S. Chem. Eur. J. 2011, 17, 4774.
(38)Oliver, C. F.; Modi, S.; Sutcliffe, M. J.; Primrose, W. U.; Lian, L.-Y.; Robert, G. C. K. Biochemistry 1997, 36, 1567.
(39)Miura, Y.; Fulco, A. J. Biochim. Biophys. Acta 1975, 388, 305.
(40)Shirane, N.; Peterson, Z. S. J. A.; Montellano, P. R. O. d. Biochemistry 1993, 32, 13732.
(41)Ravichandran, K.; Boddupalli, S.; Hasermann, C.; Peterson, J.; Deisenhofer, J. Science 1993, 261, 731.
(42)Ost, T. W. B.; Munro, A. W.; Mowat, C. G.; Taylor, P. R.; Pesseguiero, A.; Fulco, A. J.; Cho, A. K.; Cheesman, M. A.; Walkinshaw, M. D.; Chapman, S. K. Biochemistry 2001, 40, 13430.
(43)Cryle, M. J.; De Voss, J. J. Tetrahedron: Asymmetry 2007, 18, 547.
(44)Cryle, M. J.; Matovic, N. J.; De Voss, J. J. Tetrahedron Lett. 2007, 48, 133.
(45)Cryle, M. J.; De Voss, J. J. Chembiochem : a European journal of chemical biology 2008, 9, 261.
(46)Zilly, F. E.; Acevedo, J. P.; Augustyniak, W.; Deege, A.; Hausig, U. W.; Reetz, M. T. Angewandte Chemie 2011, 50, 2720.
(47)Kawakami, N.; Shoji, O.; Watanabe, Y. Angew. Chem. Int. Ed. 2011, 50, 5315.
(48)Peters, M. W.; Meinhold, P.; Glieder, A.; Arnold, F. H. J. Am. Chem. Soc. 2003, 125, 13442.
(49)Chan, S. I.; Lu, Y. J.; Nagababu, P.; Maji, S.; Hung, M. C.; Lee, M. M.; Hsu, I. J.; Minh, P. D.; Lai, J. C.; Ng, K. Y.; Ramalingam, S.; Yu, S. S.; Chan, M. K. Angew. Chem. Int. Ed. 2013, 52, 3731.
(50)Appel, D.; Lutz-Wahl, S.; Schmid, R. D.; Fischer, P. J. Biotech. 2001, 88, 167.
(51)Muller, K.; Faeh, C.; Diederich, F. Science 2007, 317, 1881.
(52)Bondi, A. J. Phys. Chem. 1964, 68, 441.
(53)Smart, B. E. J. Fluor. Chem. 2001, 109, 3.
(54)Kirsch, P. Modern Fluoroorganic Chemistry, 2004.
(55)O'Hagan, D. Chem. Soc. Rev. 2008, 37, 308.
(56)Dalvi, V. H.; Rossky, P. J. Proc. Natl. Acad. Sci. U. S. A 2010, 107, 13603.
(57)Mecinovic, J.; Snyder, P. W.; Mirica, K. A.; Bai, S.; Mack, E. T.; Kwant, R. L.; Moustakas, D. T.; Heroux, A.; Whitesides, G. M. J. Am. Chem. Soc. 2011, 133, 14017.
(58)Pongdee, R.; Liu, H. W. Bioorg. Chem. 2004, 32, 393.
(59)Blanksby, S. J.; ELLISON, G. B. Acc. Chem. Res. 2003, 36, 255.
(60)Smart, B. E.; Tatlow, J. C. New York, 1994.
(61)Chambers, R. D. Fluorine in Organic Chemistry; Blackwell: Oxford, 2004.
(62)Umemoto, T. Chem. Rev. 1996, 96, 1757.
(63)Wilkinson, J. A. Chem. Rev. 1992, 92, 505.
(64)Scott D. Taylor, C. C. K., Gabriel Hum Tetrahedron 1999, 55, 12431.
(65)Singh, R. P. S., Jean'ne M. Synthesis 2002, 2561.
(66)Dax, K.; Albert, M. In Springer-Verlag Berlin : Berlin 2001; Vol. 215, p 193.
(67)Wang, C.-L. Org. React. 1985, 34, 319.
(68)Boswell, G. A.; Ripka, W. C.; Scribner, R. M.; Tullock, C. W. New York, 1974; Vol. 21.
(69)Tozer, M. J.; Herpin, T. F. Tetrahedron 1996, 52, 8619.
(70)Dmowski, W. J. Fluor. Chem. 1986, 32, 255.
(71)Lal, G. S.; Pez, G. P.; Pesaresi, R. J.; Prozonic, F. M.; Cheng, H. J. Org. Chem 1999, 64, 7048.
(72)Bosch, P.; Camps, F.; Chamorro, E.; Gasol, V.; Guerrero, A. tetrahedron lett. 1987, 28, 4733.
(73)Yoneda, A.; Fukuhara, T.; Hara, S. Chem. commun. 2005, 3589.
(74)Bérubé, M.; Kamal, F.; Roy, J.; Poirier, D. Synthesis 2006, 18, 3085.
(75)Wang, X.; Ye, Y.; Zhang, S.; Feng, J.; Xu, Y.; Zhang, Y.; Wang, J. J. Am. Chem. Soc. 2011, 133, 16410.
(76)Parsons, A. T.; Buchwald, S. L. Angew. Chem. Int. Ed. 2011, 50, 9120.
(77)Wu, F.-L.; Ross, B. P.; McGeary, R. P. Eur. J. Org. Chem. 2010, 1989.
(78)Gan, Y.; Wang, P.; Spencer, T. A. J. Org. Chem. 2006, 71, 9487.
(79)Bérubé, M. K., Fatima; Roy, Jenny; Poirier, Donald Synthesis 2006, 18, 3085.
(80)Qiu, X.; Ong, S.; Bernal, C.; Rhee, D.; Pidgeon, C. J. Org. Chem 1994, 59, 537.
(81)Eisenberger, P.; Gischig, S.; Togni, A. Chemistry 2006, 12, 2579.
(82)Wu, F.-L.; Ross, B. P.; McGeary, R. P. Eur. J. Org. Chem. 2010, 2010, 1989.
(83)Yonghong Gan , P. W., and Thomas A. Spencer J. Org. Chem 2006, 71, 9487.
(84)Parsons, A. T.; Buchwald, S. L. Angew. Chem. Int. Ed. 2011, 50, 9120.
(85)Gu, C.; Wysocki, V. H.; Harada, A.; Takaya, H.; Kumadaki, I. J. Am. Chem. Soc. 1999, 121, 10554.
(86)Meinhold, P.; Peters, M. W.; Hartwick, A.; Hernandez, A. R.; Arnold, F. H.
Adv. Synth. Catal. 2006, 348, 763.
(87)Maurer, S. C.; Schulze, H.; Schmid, R. D.; Urlacher, V. Adv. Synth. Catal. 2003, 345, 802.
(88)Omura, T.; Sato, R. J. Biol. Chem. 1964, 239.
(89)Truan, G.; Komandla, M. R.; Falck, J. R.; Peterson, J. A. Arch. Biochem. Biophys. 1999, 366, 192.
(90)De Montellano, P. R. O. In Cytochrome P450: Structure, Mechanism, and Biochemistry, New York, 1995.
(91)Miura, Y.; Fulco, A. J. J. Biol. Chem. 1974, 249, 1880.
(92)Appel, D.; Lutz-Wahl, S.; Fischer, P.; Schwaneberg, U.; Schmid, R. D. J. Biotechnol. 2001, 88, 167.
(93)Tran, N. H.; Huynh, N.; Chavez, G.; Nguyen, A.; Dwaraknath, S.; Nguyen, T. A.; Nguyen, M.; Cheruzel, L. J, Inorg. Biochem. 2012, 115, 50.
(94)Chenprakhon, P.; Sucharitakul, J.; Panijpan, B.; Chaiyen, P. J. Chem. Edu. 2010, 87, 829.
(95)Huang, W. C.; Westlake, A. C.; Marechal, J. D.; Joyce, M. G.; Moody, P. C.; Roberts, G. C. J. Mol. Biol. 2007, 373, 633.
(96)Neeli, R.; Girvan, H. M.; Lawrence, A.; Warren, M. J.; Leys, D.; Scrutton, N. S.; Munro, A. W. FEBS lett. 2005, 579, 5582.
(97)Girvan, H. M.; Marshall, K. R.; Lawson, R. J.; Leys, D.; Joyce, M. G.; Clarkson, J.; Smith, W. E.; Cheesman, M. R.; Munro, A. W. J. Biol. Chem. 2004, 279, 23274.
(98)Chan, S. I.; Chien, C. Y. C.; Yu, C. S. C.; Nagababu, P.; Maji, S.; Chen, P. P. Y.
J. Catal. 2012, 293, 186.
(99)Nagababu, P.; Maji, S.; Kumar, M. P.; Chen, P. P. Y.; Yu, S. S. F.; Chan, S. I.
Adv. Synth. Catal. 2012, 354, 3275.

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