(R)-Phenylephrine [(R)-PE] 是一種α1腎上腺素受體興奮劑，相較於其他類腎上腺素藥物其副作用較低，目前被廣泛用於舒緩鼻充血和延長麻醉時效。利用化學合成方式生產(R)-PE時，非藥用(S)-PE幾乎占整體產量的一半，且生產過程需用到觸媒並在高溫高壓下進行，不僅成本高且對環境不友善。近期中興大學許文輝教授及楊明德教授實驗室從Serratia marcescens BCRC 109485中分離出一株具有可以將1-(3-hydroxyphenyl)-2-(methylamino) ethanone (HPMAE) 轉換成(R)-PE的short-chain dehydrogenase/reductase (SDR)，稱之為SmSDR。從SmSDR的轉換結果中發現鏡像異構物選擇性[(R)-PE)]高達99%，但整體產率及轉換率不佳，無法達到工業化應用的標準。此篇研究即是與中興大學合作，希望利用晶體繞射學技術解出SmSDR的蛋白質結構，之後再更進一步分析其結構特性、催化機制以及反應物專一性，並依據這些資訊來設計定點突變，即是利用蛋白質工程的方式改良SmSDR的催化效率使之有潛力應用在藥物生產工業上。首先我們成功的培養出SmSDR晶體並解出具有1.47Å高解析度的蛋白質結構，接著用電腦模擬的方式建立出蛋白質與反應物的作用關係，依據這些資訊來設計突變點，最後得到具有不改變鏡像選擇性且酵素活性提高的雙點突變株SmSDR-F98YF202L，且在生物轉化過程中提高了(R)-PE)的轉換率，證明了有效的突變位點的確可以使SmSDR的催化效力增加，而我們的最終目標為將SmSDR之產率及轉換率改良到足以應用在工業化生產製程上。

(R)-Phenylephrine [(R)-PE] is an α1-adrenergic receptor agonist widely used as a nasal decongestant and a cardiac agent without major side effects opposing to other adrenergic drugs such as ephedrine. In addition, the current mass-production procedure usually consists of (S) chiral form (50%). In an end to increase the specificity, a bio-catalytic transformation procedure using a novel short-chain dehydrogenase/reductase (SDR) from Serratia marcescens BCRC 10948 (Peng, G. J. et al.) to convert 1-(3-hydroxyphenyl)-2-(methylamino) ethanone (HPMAE) into an enantioseletive (R)-PE (more than 99%) has been attempted. However, this method performs relatively low conversion yield and productivity. In this study, we aim to determine the crystallographic structure of SmSDR as a structural basis to engineer high-activity SmSDR variants. Here, we report the 1.47 Å atomic-resolution apo-form structure. A liganded complex was built using Discovery Studio. Several mutants were predicted and characterized based on a structure-guided approach. A double mutant SmSDR-F98YF202L was found to display the highest activity. Furthermore, this mutant demonstrated a much higher conversion yield and productivity in the whole-cell assay, suggesting a valuable engineered variant for pharmaceutical applications.

中文摘要 I
Abstract II
Abbreviation III
List of Figures VII
List of Tables VIII
1. Introduction 1
1.1 Enzyme biocatalysis and enzyme engineering 1
1.2 Adrenergic drugs 2
1.3 Serratia marcescens 3
1.4 Short-chain dehydrogenase/reductase 4
1.5 Preliminary results on SmSDR 5
1.6 Purpose and Specific aims 5
2. Material and Methods 6
2.1 Cloning and site-directed mutagenesis of SmSDRs 6
2.2 Expression, purification and condensation of SmSDRs 6
2.3 Crystallization screening of SmSDR 8
2.4 Optimization of SmSDR crystallization conditions 8
2.5 X-ray diffraction and data processing 9
2.6 Model building and refinement of SmSDR 9
2.7 Sequence alignment, structure comparison and homologous modeling 10
2.8 Molecular simulation analysis of SmSDR-ligand complexes 10
2.9 Determination optimal activity assay conditions of SmSDR 11
2.10 Enzyme kinetics analysis of SmSDRs 12
2.11 Whole-cell biocatalysis of (R)-PE 12
2.12 High Performance Liquid Chromatography (HPLC) analysis 13
3. Results 14
3.1 Expression and purification of SmSDRs 14
3.2 Crystallization of SmSDR and SmSDR-HPMAE-NADPH ternary complex 14
3.3 X-ray diffraction and data processing 15
3.4 Structure model building, refinement and validation 15
3.5 The overall apo form 3D structure of SmSDR 16
3.6 Structural analysis of conserved residues in SDRs. 16
3.7 Establish SmSDR-HPMAE-NADPH ternary complex model 17
3.8 Design site-directed mutagenesis variants of SmSDR 18
3.9 Determination optimal activity assay conditions of SmSDR 19
3.10 Relative enzyme activity of SmSDRs 20
3.11 Enzyme kinetics analysis of SmSDRs 20
3.12 Whole-cell biocatalysis of PE produce from HPMAE by E. coli BL21 (DE3) expressing SmSDR 21
3.13 Chirality of PE produced from HPMAE by E. coli BL21 (DE3) expressing SmSDR 21
4. Discussion 22
4.1 Co-crystallization of SmSDR-HPMAE-NADPH ternary complex 22
4.2 The concepts of designing SmSDR variants 22
4.3 The catalytic properties of SmSDR 24
4.4 An alternative strategy to increase SmSDR activity 25
4.5 Current status about (R)-PE by enzyme biocatalytic method 25
4.6 Conclusion and future direction 26
5. References 27
6. Figures and Tables 33

1. Hutt, A.J. and S.C. Tan, Drug chirality and its clinical significance. Drugs, 1996. 52 Suppl 5: p. 1-12.
2. Klinger, F.D., Wolter, L., Dietrich, W., Method for preparing of L-phenylephrine hydrochloride. 2001. US 6,187,956 B1.
3. Kumar, P., Naidu, V., Gupta, P., Application of hydrolytic kinetic resolution (HKR) in the synthesis of bioactive compounds. Tetrahedron, 2007. 63(13): p. 2745-2785.
4. Pandey, R.K., Upadhyay, P.K., Kumar, P., Enantioselective synthesis of (R)-phenylephrine hydrochloride. Tetrahedron Letters, 2003. 44: p. 6245-6246.
5. Wohlgemuth, R., Biocatalysis--key to sustainable industrial chemistry. Curr Opin Biotechnol, 2010. 21(6): p. 713-24.
6. Nestl, B.M., B.A. Nebel, and B. Hauer, Recent progress in industrial biocatalysis. Curr Opin Chem Biol, 2011. 15(2): p. 187-93.
7. Huisman, G.W. and S.J. Collier, On the development of new biocatalytic processes for practical pharmaceutical synthesis. Curr Opin Chem Biol, 2013. 17(2): p. 284-92.
8. Tomsho, J.W., A. Pal, D.G. Hall, and S.J. Benkovic, Ring Structure and Aromatic Substituent Effects on the pK a of the Benzoxaborole Pharmacophore. ACS Med Chem Lett, 2012. 3(1): p. 48-52.
9. Aldridge, S., Industry backs biocatalysis for greener manufacturing. Nat Biotechnol, 2013. 31(2): p. 95-6.
10. Lutz, S., L. Liu, and Y. Liu, Engineering Kinases to Phosphorylate Nucleoside Analogs for Antiviral and Cancer Therapy. Chimia (Aarau), 2009. 63(11): p. 737-744.
11. Dincer A, T.A., Improving the stability of cellulase by immobilization on modified polyvinyl alcohol coated chitosan beads. Journal of Molecular Catalysis B: Enzymatic, 2007. 45(1-2): p. 10-14.
12. Elleuche, S., C. Schroder, K. Sahm, and G. Antranikian, Extremozymes--biocatalysts with unique properties from extremophilic microorganisms. Curr Opin Biotechnol, 2014. 29: p. 116-23.
13. Medicinal Chemistry of Adrenergics and Cholinergics.
14. Ahlquist, R.P., Present state of alpha and beta adrenergic drugs III. Beta blocking agents. Am Heart J, 1977. 93(1): p. 117-20.
15. Eisenach, J.C., M. De Kock, and W. Klimscha, alpha(2)-adrenergic agonists for regional anesthesia. A clinical review of clonidine (1984-1995). Anesthesiology, 1996. 85(3): p. 655-74.
16. Vansal, S.S. and D.R. Feller, Direct effects of ephedrine isomers on human beta-adrenergic receptor subtypes. Biochem Pharmacol, 1999. 58(5): p. 807-10.
17. Astrup, A., Thermogenic drugs as a strategy for treatment of obesity. Endocrine, 2000. 13(2): p. 207-12.
18. Loyd, V.A., et al, Handbook of nonprescription drugs, 13th ed. American Pharmaceutical Association, Washington, DC. 2002.
19. Gilman, A.G., Goodman, L.S., Hardman, J.G., Limbird, L.E., Goodman & Gilman's the pharmacological basis of therapeutics, 10th ed. McGraw-Hill Press, New York, NY.F. 2001.
20. Westfall, T.C., Westfall, D.P., Adrenergic agonists and antagonists. In: Brunton, L.L., Lazo, J.S., Parker, K.L. (Eds.), Goodman and Gilman’s the Pharmacological Basis of Therapeutics. , 11th ed. McGraw-Hill, New York.F. 2006.
21. Hanna, P.E., Adrenergic agents. In: Delgado, J.N., Remers, W.A. (Eds.), Wilson and Gisvold’s Textbook of Organic Medicinal Pharmaceutical Chemistry. , 9th ed. Lippincott-Raven, Philadelphia. 1991.
22. Sakuraba, S.T., Hisashi; Takeda, Hideo; Achiwa, Kazuo, Chem. Pharm. Bull., 1995. 43: p. 738-747.
23. Takeda, H., T. Tachinami, M. Aburatani, H. Takahashi, T. Morimoto, and K. Achiwa, Practical asymmetric synthesis of (R)-(−)-phenylephrine hydrochloride catalyzed by (2R,4R)-MCCPM-rhodium complex. Tetrahedron Letters, 1989. 30(3): p. 367-370.
24. Miyamura, Y., Process for producing L-asparaginase. US 3,627,639, 1971.
25. Subathra, D.C., S. Alam, S.K. Nag, N.S. Jemimah, V. Mohanasrinivasan, and B. Vaishnavi, Studies on growth kinetics of Serratia marcescens VITSD2 and optimization of fermentation conditions for serratiopeptidase production. Antiinflamm Antiallergy Agents Med Chem, 2014. 13(2): p. 88-92.
26. Li, X., X. Jin, X. Lu, F. Chu, J. Shen, Y. Ma, M. Liu, and J. Zhu, Construction and characterization of a thermostable whole-cell chitinolytic enzyme using yeast surface display. World J Microbiol Biotechnol, 2014. 30(10): p. 2577-85.
27. Wang, N.Q., J. Sun, J. Huang, and P. Wang, Cloning, expression, and directed evolution of carbonyl reductase from Leifsonia xyli HS0904 with enhanced catalytic efficiency. Appl Microbiol Biotechnol, 2014. 98(20): p. 8591-601.
28. Bai, F., L. Dai, J. Fan, N. Truong, B. Rao, L. Zhang, and Y. Shen, Engineered Serratia marcescens for efficient (3R)-acetoin and (2R,3R)-2,3-butanediol production. J Ind Microbiol Biotechnol, 2015. 42(5): p. 779-86.
29. Jornvall, H., M. Persson, and J. Jeffery, Alcohol and polyol dehydrogenases are both divided into two protein types, and structural properties cross-relate the different enzyme activities within each type. Proc Natl Acad Sci U S A, 1981. 78(7): p. 4226-30.
30. Persson, B. and Y. Kallberg, Classification and nomenclature of the superfamily of short-chain dehydrogenases/reductases (SDRs). Chem Biol Interact, 2013. 202(1-3): p. 111-5.
31. Filling, C., K.D. Berndt, J. Benach, S. Knapp, T. Prozorovski, E. Nordling, R. Ladenstein, H. Jornvall, and U. Oppermann, Critical residues for structure and catalysis in short-chain dehydrogenases/reductases. J Biol Chem, 2002. 277(28): p. 25677-84.
32. Oppermann, U.C., C. Filling, K.D. Berndt, B. Persson, J. Benach, R. Ladenstein, and H. Jornvall, Active site directed mutagenesis of 3 beta/17 beta-hydroxysteroid dehydrogenase establishes differential effects on short-chain dehydrogenase/reductase reactions. Biochemistry, 1997. 36(1): p. 34-40.
33. Oppermann, U., C. Filling, M. Hult, N. Shafqat, X. Wu, M. Lindh, J. Shafqat, E. Nordling, Y. Kallberg, B. Persson, and H. Jornvall, Short-chain dehydrogenases/reductases (SDR): the 2002 update. Chem Biol Interact, 2003. 143-144: p. 247-53.
34. Ghosh, D., M. Sawicki, V. Pletnev, M. Erman, S. Ohno, S. Nakajin, and W.L. Duax, Porcine carbonyl reductase. structural basis for a functional monomer in short chain dehydrogenases/reductases. J Biol Chem, 2001. 276(21): p. 18457-63.
35. Hedlund, J., H. Jornvall, and B. Persson, Subdivision of the MDR superfamily of medium-chain dehydrogenases/reductases through iterative hidden Markov model refinement. BMC Bioinformatics, 2010. 11: p. 534.
36. Zhang, R., Y. Geng, Y. Xu, W. Zhang, S. Wang, and R. Xiao, Carbonyl reductase SCRII from Candida parapsilosis catalyzes anti-Prelog reaction to (S)-1-phenyl-1,2-ethanediol with absolute stereochemical selectivity. Bioresour Technol, 2011. 102(2): p. 483-9.
37. Zilbeyaz, K. and E.B. Kurbanoglu, Highly enantiomeric reduction of acetophenone and its derivatives by locally isolated Rhodotorula glutinis. Chirality, 2010. 22(9): p. 849-54.
38. Peng, G.J., Y.C. Kuan, H.Y. Chou, T.K. Fu, J.S. Lin, W.H. Hsu, and M.T. Yang, Stereoselective synthesis of (R)-phenylephrine using recombinant Escherichia coli cells expressing a novel short-chain dehydrogenase/reductase gene from Serratia marcescens BCRC 10948. J Biotechnol, 2014. 170: p. 6-9.
39. Ho, S.N., H.D. Hunt, R.M. Horton, J.K. Pullen, and L.R. Pease, Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene, 1989. 77(1): p. 51-9.
40. Dutta, D., S. Bhattacharyya, A. Roychowdhury, R. Biswas, and A.K. Das, Crystal structure of hexanoyl-CoA bound to beta-ketoacyl reductase FabG4 of Mycobacterium tuberculosis. Biochem J, 2013. 450(1): p. 127-39.
41. Reiss, P.D., P.F. Zuurendonk, and R.L. Veech, Measurement of tissue purine, pyrimidine, and other nucleotides by radial compression high-performance liquid chromatography. Anal Biochem, 1984. 140(1): p. 162-71.
42. Lundquist, R. and B.M. Olivera, Pyridine nucleotide metabolism in Escherichia coli. I. Exponential growth. J Biol Chem, 1971. 246(4): p. 1107-16.
43. Niefind, K., J. Muller, B. Riebel, W. Hummel, and D. Schomburg, The crystal structure of R-specific alcohol dehydrogenase from Lactobacillus brevis suggests the structural basis of its metal dependency. J Mol Biol, 2003. 327(2): p. 317-28.
44. Zhang, L., Q. Xu, X. Peng, B. Xu, Y. Wu, Y. Yang, S. Sun, K. Hu, and Y. Shen, Cloning, expression and characterization of glycerol dehydrogenase involved in 2,3-butanediol formation in Serratia marcescens H30. J Ind Microbiol Biotechnol, 2014. 41(9): p. 1319-27.
45. Huisman GW, L.J., Krebber A., Practical chiral alcohol manufacture using ketoreductases. Current Opinion in Chemical Biology, 2010. 14(2): p. 122-129.
46. Reetz, M.T., P. Soni, L. Fernandez, Y. Gumulya, and J.D. Carballeira, Increasing the stability of an enzyme toward hostile organic solvents by directed evolution based on iterative saturation mutagenesis using the B-FIT method. Chem Commun (Camb), 2010. 46(45): p. 8657-8.
47. Vazquez-Figueroa, E., V. Yeh, J.M. Broering, J.F. Chaparro-Riggers, and A.S. Bommarius, Thermostable variants constructed via the structure-guided consensus method also show increased stability in salts solutions and homogeneous aqueous-organic media. Protein Eng Des Sel, 2008. 21(11): p. 673-80.
48. Huang, L., Ma, H.-M., Yu, H.-L. and Xu, J.-H., Altering the Substrate Specificity of Reductase CgKR1 from Candida glabrata by Protein Engineering for Bioreduction of Aromatic α-Keto Esters. Adv. Synth. Catal., 2014. 356(1943–1948).
49. Michael Breuer, A.P., Bernhard Hauer, Wolfgang Siegel, Method for producing L-phenylephrine using an alcohol dehydrogenase of Aromatoleum Aromaticum EBN1 (azoarcus sp. EBNl). 2013. US 8,617,854 B2