Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21781
標題: 提升 Brevibacillus agri NCHU1002 Dihydropyrimidinase 之光學選擇性俾應用於生產 L-Homophenylalanine
Enantioselective Improvement of Dihydropyrimidinase from Brevibacillus agri NCHU1002 for L-Homophenylalanine Production
作者: 羅肇凱
Lo, Chao-Kai
關鍵字: dihydropyrimidinase;hydantoinase;L-N-carbamoylase;Brevibacillus agri NCHU1002;D,L-homophenylalanylhydantoin;L-homophenylalanine
出版社: 分子生物學研究所
引用: 1. Abendroth, J., K. Niefind, O. May, M. Siemann, C. Syldatk, and D. Schomburg. 2002. The structure of L-hydantoinase from Arthobacter aurescens leads to an understanding of dihydropyrimidinase substrate and enantio specificity. Biochemistry 41:8589-97. 2. Abendroth, J., K. Niefind, and D. Schomburg. 2002. X-ray structure of a dihydropyrimidinase from Thermus sp. at 1.3 A resolution. J Mol Biol 320:143-56. 3. Albery, W. J., and J. R. Knowled. 1986. Energetics and mechanism of proline racemase. Biochemistry 25:2572-7. 4. Altenbuchner, J., M. Siemann-Herzberg, and C. Syldatk. 2001. Hydantoinases and related enzymes as biocatalysts for the synthesis of unnatural chiral amino acids. Curr Opin Biotechnol 12:559-63. 5. Ashiuchi, M., K. Soda, and H. Misono. 1999. Characterization of yrpC gene product of Bacillus subtilis IFO 3336 as glutamate racemase isozyme. Biosci Biotechnol Biochem 63:792-8. 6. Beuth, B., K. Niefind, and D. Schomburg. 2003. Crystal structure of creatininase from Pseudomonas putida: a novel fold and a case of convergent evolution. J Mol Biol 332:287-301. 7. Chao, Y. P., H. Fu, T. E. Lo, P. T. Chen, and J. J. Wang. 1999. One-step production of D-p-hydroxyphenylglycine by recombinant Escherichia coli strains. Biotechnol Prog 15:1039-45. 8. Chen, S. T., W. H. Huang, and K. T. Wang. 1994. Kinetic resolution of esters of amino acids in t-butanol containing 5% water catalyzed by a stable industrial alkaline protease. Chirality 6:572-576. 9. Cheon, Y. H., H. S. Kim, K. H. Han, J. Abendroth, K. Niefind, D. Schomburg, J. Wang, and Y. Kim. 2002. Crystal structure of D-hydantoinase from Bacillus stearothermophilus: insight into the stereochemistry of enantioselectivity. Biochemistry 41:9410-7. 10. Cheon, Y. H., H. S. Park, J. H. Kim, Y. Kim, and H. S. Kim. 2004. Manipulation of the active site loops of D-hydantoinase, a (beta/alpha)8-barrel protein, for modulation of the substrate specificity. Biochemistry 43:7413-20. 11. Cheon, Y. H., H. S. Park, S. C. Lee, and H. S. Kim. 2003. Structure-based mutational analysis of the active site residues of D-hydantoinase. J Mol Catal B: Enzymatic 26:217-222. 12. Cho, B. K., J. H. Seo, T. W. Kang, and B. G. Kim. 2003. Asymmetric synthesis of L-homophenylalanine by equilibrium-shift using recombinant aromatic L-amino acid transaminase. Biotechnol Bioeng 83:226-34. 13. Cushman, D. W., and M. A. Ondetti. 1999. Design of angiotensin converting enzyme inhibitors. Nat. Med. 5:1110-1113. 14. Flint, L. 2004. The role of ACE inhibitor therapy in treating cardiovascular disease. Nurs Times 100:34-7. 15. Gokhale, D. V., K. B. Bastawde, S. G. Patil, U. R. Kalkote, R. R. Joshi, R. A. Joshi, T. Ravindranathan, B. G. Gaikwad, V. V. Jogdand, and S. Nene. 1996. Chemoenzymatic synthesis of D(-)phenylglycine using hydantoinase of Pseudomonas desmolyticum resting cells. Enzyme Microb Technol 18:353-7. 16. Gross, C., C. Syldatk, V. Mackowiak, and F. Wagner. 1990. Production of L-tryptophan from D,L-5-indolylmethylhydantoin by resting cells of a mutant of Arthrobacter species (DSM 3747). J Biotechnol 14:363-75. 17. Houng, J. Y., and C. L. Hsieh. 1995. Method for preparing optically active homophenylalanine and esters thereof using lipase from wheat germ or Candida lipolytica. United States Patent 5,552,317. 18. Houng, J. Y., M. L. Wu, and S. T. Chen. 1996. Kinetic resolution of amino acid esters catalyzed by lipases. Chirality 8:418-22. 19. Hu, H. Y., W. H. Hsu, and H. R. Chien. 2003. Characterization and phylogenetic analysis of a thermostable N-carbamoyl- l-amino acid amidohydrolase from Bacillus kaustophilus CCRC11223. Arch Microbiol 179:250-7. 20. Ishikawa, T., K. Watabe, Y. Mukohara, and H. Nakamura. 1997. Mechanism of stereospecific conversion of DL-5-substituted hydantoins to the corresponding L-amino acids by Pseudomonas sp. strain NS671. Biosci Biotechnol Biochem 61:185-7. 21. Kao, C. H., and W. H. Hsu. 2003. A gene cluster involved in pyrimidine reductive catabolism from Brevibacillus agri NCHU1002. Biochem Biophys Res Commun 303:848-54. 22. Kim, G. J., and H. S. Kim. 1998. C-terminal regions of D-hydantoinases are nonessential for catalysis, but affect the oligomeric structure. Biochem Biophys Res Commun 243:96-100. 23. Li, X., C. H. Yeung, A. S. C. Chan, T. S. Lee, and T. K. Yang. 1999. An efficient synthesis of chiral homophenylalanine derivatives via enantioselective hydrogenation. Tetrahedron: Asymmetry 10:3863-3867. 24. Lieberman, I., and A. Kornberg. 1954. Enzymatic synthesis and breakdown of a pyrimidine, orotic acid. I. Dihydroortic acid, ureidosuccinic acid, and 5-carboxymethylhydantoin. J Biol Chem 207:911-24. 25. Liu, L., K. Iwata, M. Yohda, and K. Miki. 2002. Structural insight into gene duplication, gene fusion and domain swapping in the evolution of PLP-independent amino acid racemases. FEBS Lett 528:114-8. 26. Lo, H. H., S. K. Hsu, W. D. Lin, N. L. Chan, and W. H. Hsu. 2005. Asymmetrical synthesis of L-homophenylalanine using engineered Escherichia coli aspartate aminotransferase. Biotechnol Prog 21:411-5. 27. Lo, H. H., C. H. Kao, D. S. Lee, T. K. Yang, and W. H. Hsu. 2003. Enantioselective synthesis of (S)-2-amino-4-phenylbutanoic acid by the hydantoinase method. Chirality 15:699-702. 28. Martinez-Rodriguez, S., F. J. Las Heras-Vazquez, J. M. Clemente-Jimenez, L. Mingorance-Cazorla, and F. Rodriguez-Vico. 2002. Complete conversion of D,L-5-monosubstituted hydantoins with a low velocity of chemical racemization into D-amino acids using whole cells of recombinant Escherichia coli. Biotechnol Prog 18:1201-6. 29. May, O., P. T. Nguyen, and F. H. Arnold. 2000. Inverting enantioselectivity by directed evolution of hydantoinase for improved production of L-methionine. Nat Biotechnol 18:317-20. 30. May, O., M. Siemann, M. Pietzsch, M. Kiess, R. Mattes, and C. Syldatk. 1998. Substrate-dependent enantioselectivity of a novel hydantoinase from Arthrobacter aurescens DSM 3745: purification and characterization as new member of cyclic amidases. J Biotechnol 61:1-13. 31. May, O., M. Siemann, M. G. Siemann, and C. Syldatk. 1998. Catalytic ans structural function of zinc for the hydantoinase from Arthrobactor aurescens DSM 3745. J Mol Cata B: Enzymatic 4:211-218. 32. Patchett, A. A., E. Harris, E. W. Tristram, M. J. Wyvratt, M. T. Wu, D. Taub, E. R. Peterson, T. J. Ikeler, J. T. Broeke, L. G. Payne, D. L. Ondeyka, E. D. Thorsett, W. J. Greenlee, N. S. Lohr, R. D. Hoffsommer, H. Joshua, W. V. Ruyle, J. W. Rothrock, S. D. Aster, A. L. Maycock, F. M. Robinson, R. Hirschmann, C. S. Sweet, E. H. Ulm, D. M. Gross, T. C. Vassil, and C. A. Stone. 1980. A new class of angiotensin-converting enzyme inhibitors. Nature 288:280-283. 33. Regla, I., H. Luna, H. I. Perez, P. Demare, I. Bustos-Jaimes, and V. C. Zaldivar, M. L. 2004. Enzymic resolution of N-acetyl-homophenylalanine with mammalian kidney acetone powders. Tetrahedron: Asymmetry 15:1285-1288. 34. Samebrook, J., and D. E. Russell. 2001. Molecular cloning: a laboratory manual, 3rd ed. 35. Sharma, R., and R. M. Vohra. 1997. A thermostable D-hydantoinase isolated from a mesophilic Bacillus sp.AR9. Biochem Biophys Res Commun 234:485-8. 36. Shaw, J. P., G. A. Petsko, and D. Ringe. 1997. Determination of the structure of alanine racemase from Bacillus stearothermophilus at 1.9-A resolution. Biochemistry 36:1329-42. 37. Slavnov, V. N., V. V. Markov, V. A. Oleinik, E. V. Luchitskii, and V. M. Rudichenko. 1989. The renin-angiotensin-aldosterone system in hypertension of hypothalamic origin. Klik. Med. (Mosk) 67:60-64. 38. Syldatk, C., O. May, J. Altenbuchner, R. Mattes, and M. Siemann. 1999. Microbial hydantoinases--industrial enzymes from the origin of life? Appl Microbiol Biotechnol 51:293-309. 39. Taylor, P. P., D. P. Pantaleone, R. F. Senkpeil, and I. G. Fotheringham. 1998. Novel biosynthetic approaches to the production of unnatural amino acids using transaminases. Trends Biotechnol 16:412-8. 40. Thoden, J. B., G. N. Phillips, Jr., T. M. Neal, F. M. Raushel, and H. M. Holden. 2001. Molecular structure of dihydroorotase: a paradigm for catalysis through the use of a binuclear metal center. Biochemistry 40:6989-97. 41. Volpe, M. 2004. Hypertension therapy: mixing, matching, and meeting targets. Adv Ther 21:107-22. 42. Wang, W. C., W. C. Chiu, S. K. Hsu, C. L. Wu, C. Y. Chen, J. S. Liu, and W. H. Hsu. 2004. Structural basis for catalytic racemization and substrate specificity of an N-acylamino acid racemase homologue from Deinococcus radiodurans. J Mol Biol 342:155-69. 43. Xie, Y. L., R. Li, Z. Mi, A. Jiang, Y. 2000. DPAMPP in catalytic asymmetric reactions: enantioselective synthesis of L-homophenylalanine. Tetrahedron: Asymmetry 11:1487-1494. 44. Xu, Z., Y. Liu, Y. Yang, W. Jiang, E. Arnold, and J. Ding. 2003. Crystal structure of D-Hydantoinase from Burkholderia pickettii at a resolution of 2.7 Angstroms: insights into the molecular basis of enzyme thermostability. J Bacteriol 185:4038-49. 45. Yohda, M., H. Okada, and H. Kumagai. 1991. Molecular cloning and nucleotide sequencing of the aspartate racemase gene from lactic acid bacteria Streptococcus thermophilus. Biochim Biophys Acta 1089:234-40. 46. Zhao, H., R. G. Luo, D. Wei, and S. V. Malhotra. 2002. Concise synthesis and enzymatic resolution of L-(+)-homophenylalanine hydrocholide. Enantiomer 7:1-3. 47. 高肇鴻. 1999. Bacillus circulans D-hydantoinase 基因的選殖、表現及酵素特性. 碩士論文,分子生物研究所,中興大學,台中. 48. 高肇鴻. 2003. Brevibacillus agri NCHU1002 嘧啶還原代謝途徑的調控及 dihydropyrimidinase 應用於生產 L-homophenylalanine. 博士論文,分子生物研究所,中興大學,台中.
摘要: 
L-Homophenylalanine (L-HPA) is the precursor of anti-vessel pressure drugs. In the hydantoinase process for the production of L-HPA from homophenylalanyl-
hydantoin (HPAH), hydantoinase was coupled with L-N-carbamoylase (LNC) in the catalytic reaction. Dihydropyrimidinase from Brevibacillus agri NCHU1002 (bagHyd) has hydantoinase activity. However, enantioselectivity of bagHyd has a perference for D-HPAH. The enantioselectivity of bagHyd to L-HPAH can be increased by the addition of 5 mM and 1 mM MnCl2 in the culture medium and reaction mixture , respectively. Site-directed mutagenesis was also performed to obtain bagHyd variants with high activity toward L-HPAH. The bagHyd protein formed TIM barrel by (α/β)8 secondary structure, witch contains two metal ions in active site. Outside of the active site, three stereochemistry gate loops (SGLs) were found, in which, six residues (Met63, Phe65, Leu94, Phe152, Trp155 and Leu159) were predicted to involve in enantioselectivity to the substrate. Variants of M63A, F65A, L94A and L159A exhibited increased activity toward L-HPAH. Suggesting that the variants lead to decrease steric hindrance and facilitate the access of substrate to active site. Variant L159V with the highest activity toward L-HPAH was developed and used to convert HPAH to L-HPA in the hydantoinase process. The conversion yield of L-HPA increased to 60 % in the hydantoinase process by the replacement of L159V to wild-type bagHyd. Achieved conversion yield to 90 % in the hydantoinase process by addition of N-acylamino acid racemase (NAAAR), it decreased the accumulation of N-carbamoyl-D-HPA (NC-D-HPA). In this study, high L-HPAH activity of bagHyd variant was obtained with site-directed mutagenesis, and the conversion yield of L-HPA increased remarkably by the coupling with NAAAR in the hydantoinase process.

利用 hydantoinase搭配 L-N-carbamoylase (LNC) 可催化受質 homophenyl-
alanylhydantoin (HPAH) 生產降血壓藥物之前驅物- L-homophenylalanine (L-HPA) 。目前已自 Brevibacillus agri NCHU1002 選殖出具 HPAH 催化活性之耐熱性 dihydropyrimidinase (bagHyd) ,惟其選擇性偏向 D-HPAH 。本研究發現於培養基與反應液中分別添加 5 mM 及 1 mM 的 Mn2+ 離子時,可提升 bagHyd 對 L-HPAH 的光學選擇性。此外也利用定點突變方式改良 bagHyd ,以獲得對 L-HPAH 活性更高的變異酵素。 BagHyd 的蛋白的立體結構為 (α/β)8 二級結構所組成之桶狀結構 (TIM barrel) ,且活性中心含有金屬離子,但在其中未發現受質存在。在其活性中心外圍發現stereochemistry gate loops (SGLs) ,推測 SGLs 中六個胺基酸殘基 (Met63 、 Phe65 、 Leu94 、 Phe152 、 Trp155 及 Leu159) 可能影響受質光學選擇性,遂將上述殘基進行定點突變。分析突變酵素催化活性,結果顯示,M63A 、 L94A 、 L159A 及 F65A 變異株對 L-HPAH 之活性具明顯增加,推測此四位置胺基酸殘基變異後,藉減少受質進出障礙並協助受質進入活性中心,而增加催化活性。變異酵素中以 L159V 對 L-HPAH 的活性最高,將其應用於 hydantoinase 產程,轉換 HPAH 以生產 L-HPA。在 hydantoinase產程中以 L159V 變異酵素取代野生型 bagHyd 時,可使 L-HPA 的轉換率達到 60 % 。在產程中外加 N-acylamino acid racemase (NAAAR) 時,可降低反應中 N-carbamoyl-D-HPA (NC-D-HPA) 的累積,將 L-HPA 的轉換率提升至 90 % 。本研究中確實利用定點突變方式得到高 L-HPAH 轉換活性的變異 bagHyd ,且在 hydantoinase 產程中搭配 NAAAR 時,可將 L-HPA 的轉換率大幅提升。
URI: http://hdl.handle.net/11455/21781
其他識別: U0005-2808200614212500
Appears in Collections:分子生物學研究所

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