Title Page
Contents
LIST OF ABBREVIATIONS 16
Abstract 19
PART I. GENERAL INTRODUCTION 24
1. Inflammation in diseases. 25
2. Lipid mediators 30
3. Resolvins 32
4. Hepoxilins and trioxilins 36
5. Lipoxygenases 39
6. Purpose of this study 45
PART II. Production of two new lipid mediators from docosahexaenoic acid by combinatorial catalysis involving enzymatic and chemical reaction 47
1. MATERIALS AND METHODS 48
1.1. Materials 48
1.2. Gene cloning 49
1.3. Culture conditions and over-expression 52
1.4. Enzyme purification 53
1.5. Measurement of molecular weight and associated metal ion 54
1.6. Enzymatic activity assay 55
1.7. Enzyme kinetics measurements 56
1.8. Enzyme reactions for conversion of DHA into bioactive molecules 57
1.9. Base catalyzed hydrolysis for epoxide ring-opening 59
1.10. HPLC analysis for reactants 59
1.11. LC-MS/MS analysis for identification of reactants 63
1.12. Nuclear magnetic resonance (NMR) spectroscopy 64
1.13. Molecular modeling and substrate docking simulation 65
1.14. Database search for chemical structure 67
2. RESULTS AND DISCUSSION 68
2.1. Conserved sequence analysis 68
2.2. Preparation of Osc-LOX for enzyme assay and reaction 70
2.3. Optimal conditions for catalysis 74
2.4. Identification of metal ion in the catalytic site 77
2.5. Specific activities and kinetics against PUFAs 81
2.6. Stereopecificity and regiospecificity in the catalytic site 83
2.7. Three products generated from DHA by Osc-LOX 85
2.8. Structure identification of conversion products by Osc-LOX 92
2.9. Two products converted from 7S,15R-hydroxy-16S,17S-epoxy-docosapentaenoic acid 110
2.10. Structure identification of two products produced by hydrolysis 114
2.11. Prediction of the catalytic mechanism by 3D structure and molecular docking 125
2.12. Five lipid mediators produced by combinatorial catalysts 131
3. Conclusion 135
PART III. Production of 13-monohydroxy docosahexaenoic acid and 13R,20-dihydroxy docosahexaenoic acid by Osc-LOX Ala296Gly mutant. 138
1. Materials and methods 139
1.1. Materials 139
1.2. Mutagenesis, cloning, and expression of Osc-LOX A296G 140
1.3. Enzyme purification and measurement of molecular weight 141
1.4. Quantitative analysis of metal ions associated with catalysis 143
1.5. Effects of pH and temperature in enzyme reactions 143
1.6. Specific activity and kinetics toward five PUFAs 144
1.7. Enzyme reactions for conversion of LA and DHA into HFAs 145
1.8. HPLC analysis 146
1.9. LC-MS/MS analysis 148
1.10. Nuclear magnetic resonance (NMR) spectroscopy 149
2. Results and discussion 149
2.1. Preparation of Osc-LOX A296G mutant 150
2.2. Enzymatic characterization of Osc-LOX A296G 154
2.3. Identification of changed stereo- and regiospecificity of A296G toward LA substrate 157
2.4. Conversion of DHA into two hydroxy fatty acids by Osc-LOX A296G 161
2.5. Structural identification of two conversion products converted from DHA by Osc-LOX A296G 166
3. Conclusion 180
REFERENCES 182
ABSTRACT IN KOREAN 209
Part II 9
Table 1. Oligonucleotide Primers of osc-lox for PCR. 50
Table 2. Enzymatic activity of Osc-LOX in purification steps. 73
Table 3. The concentration of elements in Osc-LOX 78
Table 4. Specific activity and kinetic parameters of Osc-LOX. 82
Table 5. 1D NMR (¹H and ¹³C) data of 17S-HDHA. 97
Table 6. 1D NMR (¹H and ¹³C) data of resolvin D5. 100
Table 7. 1D NMR (¹H and ¹³C) data of 7S,15R-hydroxy-16S,17S-... 105
Table 8. 1D NMR (¹H and ¹³C) data of 7S,15R,16S,17S-tetrahydroxy-... 119
Table 9. List of conversion products of microalgae-derived DHA. 131
Part III 9
Table 1. Specific activity and kinetic parameters of Osc-LOX A296G. 157
Table 2. 1D NMR (¹H and ¹³C) data of 13R-hydroxy-... 171
Table 3. 1D NMR (¹H and ¹³C) data of 13R,20-diydroxy-... 176
Part I 11
Figure 1. Temporal events in a resolution of acute inflammatory response. 29
Figure 2. Classification of lipids mediators generated from PUFAs. 31
Figure 3. Pathways of docosahexaenoic acids into resolvin D series via... 34
Figure 4. Pathways of eicosapentaenoic acids into resolvin E series via... 35
Figure 5. Pathways of docosapentaenoic acids into resolvin T series via... 35
Figure 6. Pathways of polyunsaturated fatty acids into trioxilins via hepoxilins. 38
Figure 7. General catalytic mechanism of the lipoxygenases. 41
Figure 8. 3D structures of humans, plants, and bacteria LOX. 42
Part II 11
Figure 1. Synthesized sequence of osc-lox with E.coli codon optimization. 51
Figure 2. Standard curve of conversion products of DHA. 62
Figure 3. Sequence alignment analysis of the catalytic site of... 69
Figure 4. SDS-PAGE analysis and molecular weight determination of Osc-LOX. 72
Figure 5. Enzymatic properties of Osc-LOX on production of 13S-HODE. 76
Figure 6. Expression of Osc-LOX with metal ions. 80
Figure 7. Effects of metal ions on the enzymatic activity of Osc-LOX. 81
Figure 8. CP-HPLC analysis for stereo- and regiospecificity... 84
Figure 9. NP-HPLC analysis of three products generated from micro-algal... 87
Figure 10. CP-HPLC analysis for three products of Osc-LOX with DHA. 91
Figure 11. LC-MS analysis of three products generated from micro-algal DHA by Osc-LOX... 93
Figure 12. LC-MS/MS analysis of 7S,15R-hydroxy-16S,17S-epoxy-docosapentaenoic acid. 95
Figure 13. 1D NMR spectrum of 17S-HDHA. 98
Figure 14. 2D NMR spectrum of 17S-HDHA. 99
Figure 15. 1D NMR spectrum of resolvin D5 (RvD5). 101
Figure 16. 2D NMR spectrum of resolvin D5 (RvD5). 102
Figure 17. 1D NMR spectrum of 7S,15R-hydroxy-16S,17S-epoxydocosa-4Z,8E,10Z,13Z,19Z-... 106
Figure 18. 2D NMR spectrum of 7S,15R-hydroxy-16S,17S-epoxydocosa-4Z,8E,10Z,13Z,19Z-... 107
Figure 19. HSQC spectrum of two hydroxyl groups and an epoxide group of 7S,15R-hydroxy-... 108
Figure 20. NOESY spectrum of 7S,15R-hydroxy-16S,17S-epoxydocosa-4Z,8E,10Z,13Z,19Z-... 109
Figure 21. RP-HPLC analysis of epoxide ring-opened products by... 113
Figure 22. LC-MS/MS analysis of 7S,15R,16S,17S-tetrahydroxy-4Z,8E,10Z,13Z,19Z-... 116
Figure 23. 1D NMR spectrum of 7S,15R,16S,17S-tetrahydroxy-4Z,8E,10Z,13Z,19Z-... 120
Figure 24. 2D NMR spectrum of 7S,15R,16S,17S-tetrahydroxy-4Z,8E,10Z,13Z,19Z-... 121
Figure 25. HSQC spectrum of four hydroxyl groups of 7S,15R,16S,17S-tetrahydroxy-... 122
Figure 26. Changed chemical shifts were monitored in HSQC spectrum. 123
Figure 27. NOESY spectrum of 7S,15R,16S,17S-tetrahydroxy-4Z,8E,10Z,13Z,19Z-... 124
Figure 28. Complex models of lipid mediators and Osc-LOX. 126
Figure 29. Binding poses of substrate in catalytic site of Osc-LOX. 127
Figure 30. Proposed catalytic mechanism of Osc-LOX. 130
Figure 31. Conversion pathways of micro-algal DHA into lipid mediators... 132
Part III 14
Figure 1. Conserved amino acid sequence related on stereo- and... 151
Figure 2. SDS-PAGE analysis and molecular mass determination of... 153
Figure 3. Effects of pH and temperature on the production of HODE... 155
Figure 4. CP-HPLC analysis for changed regio- and stereospecificity... 160
Figure 5. NP-HPLC analysis of two products generated from DHA by... 163
Figure 6. CP-HPLC analysis to identify the stereochemistry of products... 164
Figure 7. LC-MS analysis of two products converted from DHA by Osc-... 167
Figure 8. LC-MS/MS analysis of 13R,20-dihydroxy docosahexaenoic... 168
Figure 9. 1D NMR spectrum of 13R-hydroxy-4Z,7Z,10Z,14E,16Z,19Z-docosahexaenoic acid. 172
Figure 10. 2D NMR spectrum of 13R-hydroxy-4Z,7Z,10Z,14E,16Z,19Z-docosahexaenoic acid. 173
Figure 11. HSQC spectrum of a hydroxyl groups of 13R-hydroxy-4Z,7Z,10Z,14E,16Z,19Z-... 174
Figure 12. 1D NMR spectrum of 13R,20-diydroxy-4Z,7Z,10Z,14E,16Z,18E-docosahexaenoic acid. 177
Figure 13. 2D NMR spectrum of 13R,20-diydroxy-4Z,7Z,10Z,14E,16Z,18E-docosahexaenoic acid. 178
Figure 14. HSQC spectrum of two hydroxyl groups of 13R,20-diydroxy-4Z,7Z,10Z,14E,16Z,18E-... 179