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Title Page
Contents
List of Abbreviations 6
Abstract 7
1. Introduction 9
2. Materials and Methods 16
A. Materials 16
B. Construction of CYP191A1 expression plasmid 16
C. Heterologous expression and purification of recombinant CYP191A1 18
D. Spectroscopy 18
E. Spectral binding titrations 19
F. CYP191A1 catalytic activity assays 19
1) Peroxide shunt pathway reaction of CYP191A1 20
2) Enzymatic activities through P450 catalytic cycle using type I and II redox partners 20
3) Fatty acids hydroxylation assay 20
4) Hydroxylation assay of testosterone and 17β-estradiol 21
5) Hdroxylation assay of chromogenic substrates 22
6) Oxidation assay of fluorogenic substrates 23
7) Hydroxylation of drug substrates by CYP191A1 24
3. Results 28
A. Heterologous expression of M. Smegmatis CYP191A1 in E. coli 28
B. Spectral properties of purified CYP191A1 enzyme 28
C. Spectral equilibrium binding titrations of purified CYP191A1 enzyme for substrates 28
D. Catalytic activities of CYP191A1 towards fatty acids 32
E. Catalytic activities of CYP191A1 towards steroids 36
F. Catalytic activities of CYP191A1 towards chromogenic and fluorogenic substrates 39
G. Catalytic activities of CYP191A1 towards human drug substrates 45
4. Discussion 54
5. References 56
국문초록 60
Table 1. Cytochrome P450 from M. smegmatis str. MC2 155 strain 10
Table 2. Conversion of verapamil to : (A) human metabolites, and (B) new metabolites by CYP191A1 51
Fig. 1. Schemetic representation of catalytic cycle and peroxide shunt pathway of cytochrome P450. The key step in this cycle is the insertion of one atom of molecular oxygen to the substrate, which requires... 13
Fig. 2. Electron transfer systems in cytochrome P450. Class I systems (A) comprise a P450, a ferredoxin (FD) and an FAD-containing NAD(P)H-dependent ferredoxin reductase (FDR) that channel electrons as... 14
Fig. 3. Scheme of cloning procedure of CYP191A1 into pCW vector. The PCR amplification were performed to amplify the CYP191A1 fragment from M. smegmatis.... 17
Fig. 4. Absorption spectra of purified CY P191A1 from Mycobacterium smegmatis. (A) Reduced carbon monoxide difference spectra w ere determined using purified CY P191A1.... 29
Fig. 5. Titration of ferric CYP191A1 with (A) capric acid, (B) lauric acid, (C) myristic acid, (D) palmitic acid, (E) coumarin, (F) testosterone and (G) 17β-estradiol.... 30
Fig. 6. HPLC analysis of fatty acid oxidation catalyzed by CYP191A1. The substrates included in the reaction mixture are as follows : A, capric acid; B, lauric acid; C, myristic acid; D, palmitic acid.... 33
Fig. 7. Catalytic activity of the CYP191A1 towards fatty acids. The following P450 enzyme activities of the purified CYP191A1 were characterized; the hydroxylation of fatty acids; A, capric... 34
Fig. 8. Characterization of catalytic activities of testosterone by CYP191A1. A is the reaction under H₂O₂, showing two metabolites, 7α-OH and 6β-OH testosterone.... 37
Fig. 9. Characterization of catalytic activities of 17β-estradiol by CYP191A1. The reaction of CYP191A1 under H₂O₂ (A) and t-BHP (B) show two monohydroxylated metabolites, M1 and M2.... 38
Fig. 10. Molecular structures of p-NP and its product (A), and p-NCA and its product (B). The formation of product is catalyzed by P450 enzymes.... 40
Fig. 11. Rates of products formation of p-NP and p-NCA by CYP191A1. The CYP191A1 showed the apparent catalytic activities for p-NP under CPR, PDR/Pdx and both of peroxides and for p-NCA under FDR/Fdx,... 41
Fig. 12. Rates of products formation of fluorogenic substrates by CYP191A1. The catalytic activities of CYP191A1 for coumarin and 7-EC (A), which were analyzed by HPLC, were shown.... 43
Fig. 13. molecular structures of statin substrates. The red allows indicates the possible position of hydroxylation of human metabolites. The statin drugs are used for therapy of hypertension since they act as an... 46
Fig. 14. Rates of 6'β-OH and 6'-exomethylene statin formation of simvastatin and lovastatin by CYP191A1 under both of peroxides. CYP191A1 produced two metabolites, unlikely human type P450,... 47
Fig. 15. HPLC analysis and oxidation rate of 4-OH product formation of atorvastatin by CYP191A1 under peroxide. 4-hydroxylation activities of CYP191A1 were identified by HPLC (A) and the oxidation rates of... 48
Fig. 16. HPLC analysis and oxidation rate of 6-OH product formation of chlorzoxazone by CYP191A1 under peroxide. 6-hydroxylation activities of CYP191A1 were identified by HPLC (A) and the oxidation rates of... 50
Fig. 17. HPLC analysis and oxidation rates of products formation of verapamil by CYP191A1 under peroxide. HPLC chromatogram indicates the catalytic activities of CYP191A1 under H₂O₂.... 52
Fig. 18. LC-MS elution profile of resveratrol and its metabolites produced by CYP191A1. A shows the reference sample without reductase system or peroxide, which are identified the trans- and cis- resveratrol... 53
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