Table of Contents CHAPTER 1 INTRODUCTION 1 1.1 Introduction of esterase 1 1.2 α/β hydrolase fold 2 1.3 Catalytic triad in serine protease 2 1.4 Substrate 3 CHAPTER 2 MATERIALS AND METHODS 5 2.1 Crystallization 5 2.2 Molecular Replacement 5 2.3 X-ray Data Collection and Process 5 2.4 Structure Determination and Refinement 6 CHAPTER 3 RESULTS AND DISCUSSIONS 8 3.1 EST monomer structure 8 3.2 EST active site 8 3.3 Complex structure 9 3.4 Substrate specificity 10 3.5 The entry of the pocket 11 CHAPTER 4 CONCLUSIONS 13 Reference 14 Figures and Tables 16 Appendix 29 CHAPTER 1 INTRODUCTION 30 CHAPTER 2 Materials and Methods 32 2.1 Cloning 32 2.2 Ligation 32 2.3 The purification of cinnamoyl-CoA reductase from rice 32 2.4 Crystallization 33 CHAPTER 3 Results and Discussions 34 CHAPTER 4 Conclusions 35 Reference 36 Figures and Tables 38 List of figures and tables FIGURE 1 The catalytic mechanism of serine protease 16 FIGURE 2 EST catalyzes the stereoselective hydrolysis of DL-MATI and DL-ATIA to produce DAT 17 FIGURE 3 Structure of the EST with DL-ATIA which exist in trimer form 18 FIGURE 4 α /ß canonical hydrolase fold 19 FIGURE 5 The DL-ATIA is surrounded by the hydrophobic residues 20 FIGURE 6 A comparison of b-factor lable structure 21 FIGURE 7 The real position of substrate in DL-ATIA-EST 22 FIGURE 8 Active-site overlap of PpEST and PFE 23 FIGURE 9 The slab views show that the entry of PFE and PpEST 24 FIGURE 10 The electrostatic surface of EST 25 FIGURE 11 The entry of the pocket is shown in the slab view 26 FIGURE 12 Lignin biosynthetic pathway 38 FIGURE 13 Comparison with different cycles 39 FIGURE 14 Degradation of recombinant CCR 40 FIGURE 15 The time course experiment 41 FIGURE 16 The precipitant was collected to show in SDS-PAGE 42 FIGURE 17 The recombinant and fusion-like protein after expression 43 FIGURE 18 The fraction from different peaks 44 Table 1 Crystal diffraction and structural statistics of EST 27 Table 2 Substrate specificity of EST toward linear esters 28 Table 3 PCR condition 45 Table 4 Primers 45