Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23575
標題: 探討放線菌屬kentuckense轉麩胺醯胺酶的結晶與分子結構表徵
Preliminary crystallization and structure characterization of transglutaminase from Streptoverticillium kentuckense
作者: 姜惠文
Jiang, Huei-Wen
關鍵字: 轉麩胺醯胺酶;transglutaminase
出版社: 生物化學研究所
引用: 1. Motokia, M. and K. Seguro, (1998). Transglutaminase and its use for food processing. Trends Food Sci. Technol. 9, 204-210. 2. Aeschlimann, D. and M. Paulsson, (1994). Transglutaminases: protein cross-linking enzymes in tissues and body fluids. Thromb. Haemost. 71(4), 402-415. 3. Serafini-Fracassini, D. and S. Del Duca, (2008). Transglutaminases: widespread cross-linking enzymes in plants. Annal. Bot. 102, 145-152. 4. Yokoyama, K., N. Nio, and Y. Kikuchi, (2004). Properties and applications of microbial transglutaminase. Appl. Microbiol. Biotechnol. 64(4), 447-454. 5. Zhu, Y. and J. Tramper, (2008). Novel applications for microbial transglutaminase beyond food processing. Trends Biotechnol. 26(10), 559-565. 6. Ando, H., Adachi, M., Umeda, K., Matsuura, A., and Nonaka, M., (1989). Purification and Characteristics of a Novel Transglutaminase Derived from Microorganisms. Agric. Biol. Chem. 53, 2613-2617. 7. Washizu, K., Ando, K., Koikeda, S., Hirose, S., Matsuura, A., Takagi, H., Motoki, M., and Takeuchi, K., (1994). Molecular Cloning of the Gene for Microbial Transglutaminase from Streptoverticillium and Its Expression in Streptomyces lividans. Biosci. Biotech. Biochem. 58(1), 82-87. 8. Gerber, U., Jucknischke, U., Putzien, S., and Fuchsbauer, H. L., (1994). A rapid and simple method for the purification of transglutaminase from Streptoverticillium mobaraense. Biochem. J. 299, 825-829. 9. Pasternack, R., et al., (1998). Bacterial pro-transglutaminase from Streptoverticillium mobaraense-purification, characterisation and sequence of the zymogen. Eur. J. Biochem. 257(3), 570-576. 10. Yang, M.T., Chang, C. H., Wang, J. M., Wu, T. K., Wang, Y. K., Chang, C. Y., Thomas Li, T. H., (2010). Crystal structure and inhibition studies of transglutaminase from Streptomyces mobaraense. J. Biol. Chem. 286, 7301-7307. 11. Noguchi, K., et al., (2001). Crystal structure of red sea bream transglutaminase. J. Biol. Chem. 276(15), 12055-12059. 12. Yee, V. C., et al., (1994). Three-dimensional structure of a transglutaminase: Human blood coagulation factor XIII. Proc. Nati. Acad. Sci. 91, 7296-7300. 13. Kachiwagi, T., et al., (2002). Crystal structure of microbial transglutaminase from Streptoverticillium mobaraense. J. Biol. Chem. 277(46), 44252-44268. 14. Shimba, N., K. Yokoyama, and E. Suzuki, (2002). NMR-based screening method for transglutaminases: rapid analysis of their substrate specificities and reaction rates. J. Agric. Food Chem. 50(6), 1330-1334. 15. Chang, S.C., Su, M. H., Wu Lee, Y. H., (1997). Roles of signal peptide and mature domains in the secretion and maturation of the neutral metalloprotease from Streptomyces cacaoi. Biochem. J. 321, 29-37. 16. 吳師誠. (2002). Streptomyces kentuckense轉麩氨醯胺酶基因之篩選與酵素特性之研究. 中興大學分子生物研究所碩士論文. 17. Matthews, B.W., (1968). Solvent content of protein crystals. J. Mol. Biol. 33, 491-497. 18. 江厚德. (2008). 水溶性XCC菌纖維分解酶基因選殖及表達與放線菌轉麩胺醯胺酶的純化與結晶. 中興大學生物化學研究所碩士論文. 19. CCP4(版本6.2.0)及COOT(版本0.6.2)下載網址: http://www.ccp4.ac.uk/download/os.php
摘要: 
轉麩胺醯胺酶(Transglutaminase,簡稱TGase; EC 2.3.2.13)經由醯基轉移反應,催化麩醯胺酸(glutamine)的r-羧酸基(r-carboxyamide)與各種一級胺基(如lysine的胺基)形成鍵結。解析Streptoverticillium mobaraense菌中不具活性TGase(smTGase) zymogen酶原的晶體結構,顯示位於酵素前端的pro-region以L狀構形結合在裂溝狀的活性區域上方,並以Tyr12及Tyr16抑制酵素Cys110, His320及Asp301所形成的活性催化作用區。研究顯示原核生物中分泌性蛋白質例如Streptomyces cacaoi 的metalloprotease,pro-region有助於折疊分泌後的酵素結構使具有活性的必要性。Streptoverticillium kentuckense (sk)菌屬的TGase的pro-region序列與smTGase近似,但是對應的胺基酸為Tyr12及His16。本計畫探討skTGase中pro-region結構及其抑制模式,藉以改造pro-region,在不影響酵素活性的前提下,於大量表達後進行外加酵素截切時容易分離。目前已在大腸桿菌BL21*(DE3)(pMT32C-SnPTGA)(pRK1037)中表達1.0升及500 mL的水溶性skTGase zymogen,使用Ni-NTA (Qiagen)及HiTrap-Q (Pharmacia biotech)管柱液相層析純化並濃縮至23.49 mg/mL。以蒸氣昇華法培養晶體,於國家同步輻射中心BL13B1站測得晶體繞射解析度為1.5 A。skTGase晶體的空間群為P21,單位晶格尺寸a=48.38 A、b=95.44 A、c=79.83 A、β=92.45°。由CCP4套件中Matthew’s coefficient預估,推測每個不對稱單元具有2個分子。以smTGase結構為模板,使用分子取代法求得初步電子雲圖,R值為46 %。使用COOT軟體建構分子模型並搭配CCP4套件中的REFMAC做結構最佳化,重複循環數次後R值降至41%,目前由電子雲分佈只能建構長度61-262的部份結構(全長395胺基酸),晶體不對稱單元中chain A與chain B各別完成91 %及79 %的建構。為了解析全長skTGase結構,目前仍需調整結晶實驗條件以得到高品質的晶體樣品。

Transglutaminase (TGase;EC 2.3.2.13) catalyzes an acyl transfer reaction which forms covalent bond between the r-carboxyamide group of glutamine residues of protein and a variety of primary amines, including the amino group of lysine residue. The structural analysis of smTGase zymogen from Streptoverticillium mobaraense (sm) reveals that the L-shaped pro-region at the N-terminus complements the topology of the active cleft region and two key residues, Tyr12 and Tyr16, located at the top of the catalytic triad (Cys110, Asp301, and His320) to block the enzyme activity. It is plausible that the function of the pro-region resembles that of the metalloprotease from Streptomyces cacaoi which is essential for enzyme activity on folding of the protein structure after secretion. The pro-region of skTGase from Streptoverticillium kentuckense is similar to that of smTGase but the corresponding inhibitory residues are Tyr12 and His16. We aim to investigate the crystal structure and the inhibition mode of skTGase for protein engineering in producing less sticky pro-region after enzymatic cleavage. We have expressed soluble skTGase zymogen in 1.0 and 0.5 liter culture of E. coli strain BL21*(DE3)(pMT32C-SnPTGA)(pRK1037). The enzyme was purified by using Ni-NTA (Qiagen) and HiTrap-Q (Pharmacia biotech), and concentrated to 23.49 mg/mL. The crystal were grown by hanging drop vapor diffusion method and diffracted to 1.5 A resolution limit. The skTGase crystal belongs to space group P21 with unit cell dimensions of a=48.38 A, b=95.44 A, c=79.83 A, and β=92.45°. There are two molecules per asymmetric unit based on the estimated Matthew’s coefficient. The initial phasing was solved by using the molecular replacement method and the structure of smTGase. The initial R factor was 46 % and the model was built using COOT. At present, only part of the structure, 61-262 residues out of 395 amino acids, was visible and fitted in the electron density map and the R factor is 41% after several rounds of structural refinement by using REFMAC5.0 in the suite of CCP4 program package. The completeness of the sequence fitted for chain A and chain B in the asymmetric unit were 91% and 79% , respectively. Further refining the crystallization condition to obtain a good quality crystal is necessary to solve the full-length skTGase structure.
URI: http://hdl.handle.net/11455/23575
其他識別: U0005-1207201215442700
Appears in Collections:生物化學研究所

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