臺灣博碩士論文加值系統

3α-Hydroxysteroid dehydrogenase/carbonyl reductase ( 3α-HSD/CR，EC 1.1.1.50 )是從Comamonas testosteroni菌體中分離而來的誘發性酵素，利用NAD+為輔因子，將受質androsterone氧化成androstanedione並伴隨NADH的生成而完成催化反應。3α-HSD/CR是屬於SDRs家族成員之一，其立體結構已經在2000年利用X-ray晶體繞射所解出。3α-HSD/CR在結構上為一同源雙聚體，並和其它家族成員一樣具有βαβ二級結構所疊合組成的Rossmann-fold特徵。我們從結構上分析並推測，3α-HSD/CR的雙聚體聚合化主要是發生於各單元體中C端的αCT domain上的Asp249和另一個單元體中的αF domain上的Arg167之間形成鹽橋作用力而來提供整個雙套體結構的穩定力量。為了證實這個鹽橋作用力是否就是穩定3α-HSD/CR的雙套體結構，於是便針對Asp249，利用定位突變法，分別置換成Ala、Lys及Ser，來改變Asp249和Arg167之間的交互作用力，並且利用酵素動力學、圓偏振二旋圖譜、螢光掃描、膠體過濾層析、超高速離心、熱變性和尿素變性等實驗，來探討野生型及突變型3α-HSD/CR的雙聚體結構及結構穩定度。實驗結果中，在催化活性上，D249A和D249K的kcat/Km分別下降了30倍和1416倍，而D249S和野生型相比則沒有太大差異。從二級結構上雖然無法看到wild-type和Asp-249突變種之間有明顯差異，但從螢光圖譜可發現3α-HSD/CR序列中唯一的Trp所處的環境產生了改變。從膠體過濾和超高速離心實驗結果，發現D249A、D249K和D249S都會隨著濃度改變而影響聚合化，而具有self-association現象。其中，D249A和D249K在低濃度時，主要是以單體存在。最後從熱變性和尿素變性實驗結果中發現這三株突變種的結構穩定度都比野生型還要差。因此，從以上結果總結:各單元體中Asp249和Arg167之間的鹽橋作用力確實是穩定3α-HSD/CR雙套體結構的重要力量。

3α-Hydroxysteroid dehydrogenase/carbonyl reductase ( 3α-HSD/CR，EC 1.1.1.50 ) from Comamonas testosteroni is an inducible enzyme which catalyzes the oxidation of androsterone with NAD+ to form androstanedione and NADH. 3α-HSD/CR was a member of the SDRs family, whose crystal structure had been solved by X-ray diffraction in 2000. The structure of 3α-HSD/CR is a homodimer which shows a typical pattern of Rossmann-fold structure consisting of βαβ units, and reaveals similarity with other members upon the SDRs family. The dimerization in 3α-HSD/CR had been proposed taken place via the Asp249 in helix αCT of each subunit which forms a salt-bridge interaction with the Arg167 in αF helix of the other subunit from structural analysis. To confirm this hypothesis, we performed site-specific mutagenesis to replace Asp249 to Ala, Lys and Ser, respectively, and also utilized kinetic assay, circular dichroism, fluorescence scanning, gel filtration chromatography, analytical ultracentrifugation, thermal and urea unfolding experiments to evaluate the role of Asp249 stabilizing dimer formation and maintaining structural stability in 3α-HSD/CR. In kinetic assay, kcat/Km of D249S was similar to wild-type, but kcat/Km of D249A and D249K were decreased by 30-fold and 1416-fold compared with wild-type, respectively. There were no differences in circular dichroism spectrum between wild-type and mutants, but the tertiary structure revealed variations from the fluorescence analysis. This suggests the only Trp residue in mutants of 3α-HSD/CR had been exposed to a hydrophilic environment. In the gel filtration and sedimentation velocity experimental data, we found that dimerization of wild-type and mutants exhibited a concentration-dependence. Especially D249A and D249K showed a predominately monomeric form under the low concentrations. The results from thermal and urea unfolding experiments showed that the structural stabilities of these Asp249 mutants were worse than that of wild-type. In conclusion, the salt-bridge interaction formed by Asp249 and Arg167 of each subunit were important for stabilizing dimer formation in 3α-HSD/CR.

目錄
頁次
圖目錄…………………………………………………………………… B
表目錄…………………………………………………………………… D
縮寫表…………………………………………………………………… E
中文摘要………………………………………………………………… F
英文摘要………………………………………………………………… H
壹、前言………………………………………………………………… 1
貳、實驗分法與材料
一、藥品與試劑……………………………………………………. 7
二、緩衝溶液製備…………………………………………………. 10
三、主要儀器與器材………………………………………………. 13
四、實驗方法
1. 突變型3α-HSD/CR質體 (pET15b-3α-HSD/CR)製備…… 15
2. 重組野生型及突變型3α-HSD/CR之純化…………………19
3. 酵素基本活性測定…………………………………………..23
4. 酵素動力學參數測定………………………………………..24
5. 圓偏振二旋圖譜分析………………………………………..24
6. 螢光掃描……………………………………………………..25
7. 膠體過濾層析………………………………………………..26
8. 分析級超高速離心機進行沉降速率分析…………………..27
9. 稀釋酵素隨時間增加測其活性……………………………..28
10. 蛋白質結構熱變性實驗……………………………………..30
11. 尿素破壞蛋白質結構及活性實驗
(Urea unfolding study)……………………………………….31
参、實驗結果…………………………………………………………….33
肆、討論………………………………………………………………….44
伍、參考文獻…………………………………………………………….52









圖目錄

頁次
圖1 3α-HSD/CR的單套體結合輔因子NAD+的立體結構圖……….57
圖2、3α-HSD/CR的催化反應機轉圖………………………………....58
圖3、SDRs家族成員在folding topology上的比較………………......59
圖4、SDRs家族成員立體結構上的比較……………………………...60
圖5、位於P軸交界面上兩個單元體之間的Asp249和Arg167示
意圖……………………………………………………………....61
圗6、重組野生型及突變型3α-HSD/CR在純化過程後14%
SDS-PAGE分析圖………………………………………………62
圖7、重組野生型及重組突變型 3α-HSD/CR ( 0.2 mg/ml ) 利用
Far-UV CD掃描195~240 nm的全光譜圖……………………..65
圖8、重組野生型及重組突變型 3α-HSD/CR ( 0.02 mg/ml ) 在
280 nm下利用螢光掃描發射波長300~400 nm的全光譜圖….66
圖9、重組野生型及重組突變型 3α-HSD/CR ( 0.02 mg/ml ) 在
295 nm下利用螢光掃描發射波長300~400 nm的全光譜圖….67
圖10、Low molecular weight kits ( Amersham Bioscience ) 標準品
以Superdex G75管柱進行分離後，所建立出來的標準曲線...68
圗11、重組野生型 3α-HSD/CR ( 1.0 ~ 0.01 mg/ml )以Superdex G75
管柱，0.3 ml/min流速，各取100μl進行分離..........................69
圖12、重組突變型 D249A 3α-HSD/CR ( 1.0 ~ 0.01 mg/ml )以
Superdex G75管柱，0.3 ml/min流速，各取100μl進行分離..70
圖13、重組突變型 D249K 3α-HSD/CR ( 1.0 ~ 0.01 mg/ml )以
Superdex G75管柱，0.3 ml/min流速，各取100μl進行分離..71
圖14、重組突變型 D249S 3α-HSD/CR ( 1.0 ~ 0.01 mg/ml )以
Superdex G75管柱，0.3 ml/min流速，各取100μl進行分離..72
圖15、重組野生型和重組突變型( D249A、D249K和D249S )
3α-HSD/CR在Superdex G75管柱分離綜合比較圖…………73
圖16、重組野生型3α-HSD/CR ( 1.0 mg/ml )的沉降速率分析............74
圖17、重組野生型3α-HSD/CR ( 0.05 mg/ml )的沉降速率分析..........75
圖18、重組突變型D249A 3α-HSD/CR ( 1.0 mg/ml )的沉降速率
分析..............................................................................................76
圖19、重組突變型D249A 3α-HSD/CR ( 0.02 mg/ml )的沉降速率
分析..............................................................................................77
圖20、重組突變型D249K 3α-HSD/CR ( 1.0 mg/ml )的沉降速率
分析..............................................................................................78
圖21、重組突變型D249K 3α-HSD/CR ( 0.02 mg/ml )的沉降速率
分析..............................................................................................79
圖22、重組突變型D249S 3α-HSD/CR ( 1.0 mg/ml )的沉降速率
分析..............................................................................................80
圖23、重組突變型D249S 3α-HSD/CR ( 0.02 mg/ml )的沉降速率
分析..............................................................................................81
圖24、分析重組突變型 3α-HSD/CR隨著不同蛋白質濃度，離心後
的沉降係數和分子量比較圗…………………………………..82
圖25、重組野生型及重組突變型 3α-HSD/CR先取適量蛋白質
稀釋於kinetic assay溶液隨時間變化測其活性…………........83
圖26、重組野生型及重組突變型 3α-HSD/CR ( 0.5 mg/ml )，測
量20℃~ 70℃之間，單一波長222 nm下的吸收變化............84
圖27、重組野生型3α-HSD/CR ( 0.5 mg/ml )在不同濃度的urea
中，二級結構的吸收變化及活性的變化……………………..85
圖28、重組突變型D249A 3α-HSD/CR ( 0.5 mg/ml )在不同濃度
的urea中，二級結構的吸收變化及活性的變化…………….86
圖29、重組突變型D249K 3α-HSD/CR ( 0.5 mg/ml )在不同濃度
的urea中，二級結構的吸收變化及活性的變化…………….87
圖30、重組突變型D249S 3α-HSD/CR ( 0.5 mg/ml )在不同濃度
的urea中，二級結構的吸收變化及活性的變化…………….88
圖31、重組野生型和突變型3α-HSD/CR在各種不同濃度的urea
中二級結構變性及失去活性的綜合比較圗………………......89
圖32、突變型和野生型在第249個胺基酸的重疊比較圗…………...91
















表目錄
頁次
表一、重組野生型及突變型3α-HSD/CR之純化表………………...63
表二、重組野生型和突變型3α-HSD/CR在0.1M Caps
( pH=10.00 ) 酵素動力學參數表……………………………..64
表三、重組野生型和突變型3α-HSD/CR在尿素變性實驗中的
參數整理表…………………………………………………….90

(1)Oppermann, U. C., Belai, I., and Maser, E. (1996) Antibiotic resistance and enhanced insecticide catabolism as consequences of steroid induction in the gram-negative bacterium Comamonas testosteroni. J. Steroid Biochem. Mol. Biol. 58, 217-23.

(2)Talalay, P., Dobson, M. M., and Tapley, D. F. (1952) Oxidative degradation of testosterone by adaptive enzymes. Nature 170, 620-1.

(3)Benach, J., Filling, C., Oppermann, U. C., Roversi, P., Bricogne, G., Berndt, K. D., Jornvall, H., and Ladenstein, R. (2002) Structure of bacterial 3beta/17beta-hydroxysteroid dehydrogenase at 1.2 A resolution: a model for multiple steroid recognition. Biochemistry 41, 14659-68.

(4)Oppermann, U. C., and Maser, E. (1996) Characterization of a 3 alpha-hydroxysteroid dehydrogenase/carbonyl reductase from the gram-negative bacterium Comamonas testosteroni. Eur. J. Biochem. 241, 744-9.

(5)Jornvall, H., Persson, B., Krook, M., Atrian, S., Gonzalez-Duarte, R., Jeffery, J., and Ghosh, D. (1995) Short-chain dehydrogenases/reductases (SDR). Biochemistry 34, 6003-13.

(6)Oppermann, U., Filling, C., Hult, M., Shafqat, N., Wu, X., Lindh, M., Shafqat, J., Nordling, E., Kallberg, Y., Persson, B., and Jornvall, H. (2003) Short-chain dehydrogenases/reductases (SDR): the 2002 update. Chem. Bio.l Interact. 143-144, 247-53.

(7)Xiong, G., and Maser, E. (2001) Regulation of the steroid-inducible 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase gene in Comamonas testosteroni. J. Biol. Chem. 276, 9961-70

(8)Rossmann, M. G., and Argos, P. (1975) A comparison of the heme binding pocket in globins and cytochrome b5. J. Biol. Chem. 250, 7525-32.

(9)Maser, E., Xiong, G., Grimm, C., Ficner, R., and Reuter, K. (2001) 3alpha-Hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni: biological significance, three-dimensional structure and gene regulation. Chem. Bio.l Interact. 130-132, 707-22.

(10)Grimm, C., Maser, E., Mobus, E., Klebe, G., Reuter, K., and Ficner, R. (2000) The crystal structure of 3alpha -hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni shows a novel oligomerization pattern within the short chain dehydrogenase/reductase family. J. Biol. Chem. 275, 41333-9.

(11)Ensor, C. M., and Tai, H. H. (1996) Site-directed mutagenesis of the conserved serine 138 of human placental NAD+-dependent 15-hydroxyprostaglandin dehydrogenase to an alanine results in an inactive enzyme. Biochem. Biophys. Res. Commun. 220, 330-3.

(12)Oppermann, U. C., Filling, C., Berndt, K. D., Persson, B., Benach, J., Ladenstein, R., and Jornvall, H. (1997) Active site directed mutagenesis of 3 beta/17 beta-hydroxysteroid dehydrogenase establishes differential effects on short-chain dehydrogenase/reductase reactions. Biochemistry 36, 34-40.

(13)Cols, N., Atrian, S., Benach, J., Ladenstein, R., and Gonzalez-Duarte, R. (1997) Drosophila alcohol dehydrogenase: evaluation of Ser139 site-directed mutants. FEBS Lett. 413, 191-3.

(14)Obeyesekere, V. R., Trzeciak, W. H., Li, K. X., and Krozowski, Z. S. (1998) Serines at the active site of 11 beta-hydroxysteroid dehydrogenase type I determine the rate of catalysis. Biochem. Biophys. Res. Commun. 250, 469-73.

(15)Vedadi, M., Barriault, D., Sylvestre, M., and Powlowski, J. (2000) Active site residues of cis-2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase from Comamonas testosteroni strain B-356. Biochemistry 39, 5028-34.

(16)Filling, C., Berndt, K. D., Benach, J., Knapp, S., Prozorovski, T., Nordling, E., Ladenstein, R., Jornvall, H., and Oppermann, U. (2002) Critical residues for structure and catalysis in short-chain dehydrogenases/reductases. J. Biol. Chem. 277, 25677-84.

(17)Mobus, E., and Maser, E. (1998) Molecular cloning, overexpression, and characterization of steroid-inducible 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni. A novel member of the short-chain dehydrogenase/reductase superfamily. J. Biol. Chem. 273, 30888-96.

(18)Marcus, P. I., and Talalay, P. (1956) Induction and purification of alpha- and beta-hydroxysteroid dehydrogenases. J. Bio.l Chem. 218, 661-74.

(19)Hwang, C. C., Chang, Y. H., Hsu, C. N., Hsu, H. H., Li, C. W., and Pon, H. I. (2005) Mechanistic roles of Ser-114, Tyr-155, and Lys-159 in 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni. J. Biol. Chem. 280, 3522-8.

(20)Maser, E., Mobus, E., and Xiong, G. (2000) Functional expression, purification, and characterization of 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni. Biochem. Biophys. Res. Commun. 272, 622-8.

(21)Varughese, K. I., Skinner, M. M., Whiteley, J. M., Matthews, D. A., and Xuong, N. H. (1992) Crystal structure of rat liver dihydropteridine reductase. Proc. Natl. Acad. Sci. U S A 89, 6080-4.

(22)Somers, W. S., Stahl, M. L., and Sullivan, F. X. (1998) GDP-fucose synthetase from Escherichia coli: structure of a unique member of the short-chain dehydrogenase/reductase family that catalyzes two distinct reactions at the same active site. Structure 6, 1601-12.

(23) Bradford, M. M. ( 1976) A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the
principle of protein-dye binding. Anal. Biochem. 72, 248-54.

(24) Shuck, P. (2000) Size-distribution analysis of macromolecules by
sedimentation velocity ultracentrifugation and lamm equation
modeling. Biophys. J. 78, 1606-19.

(25) Schuck, P., Perugini, M. A., Gonzales, N. R., Howlett, G. J., and
Schubert, D. (2002) Size-distribution analysis of proteins by
analytical ultracentrifugation: strategies and application to model
systems. Biophys. J. 82, 1096-11.

(26) Neet, K. E., and Timm, D. E. (1994) Conformational stability of dimeric proteins: quantitative studies by equilibrium denaturation. Protein Sci 3, 2167-74.

(27) Huang, S. M., Chou, W. Y., Lin, S. I., and Chang, G. G. (1998) Engineering of a stable mutant malic enzyme by introducing an extra ion-pair to the protein. Proteins 31, 61-73.

(28) Giampiero, M., Almerinda, D. V., Nicola, R., and Alessandro, F. A.
(2004) The importance of being dimeric. FEBS J. 272, 16-27.

(29) Monod, J., Wyman, J. and Changeux, J. P. (1965) On the nature
of allosteric transitions: A plausible model. J. Mol. Biol. 12,
88-118.

(30) Ghosh, D., Sawicki, M., Pletnev, V., Erman, M., Ohno, S., Nakajin, S., and Duax, W. L. (2001) Porcine carbonyl reductase. structural basis for a functional monomer in short chain dehydrogenases/reductases. J. Biol. Chem. 276, 18457-63.
(31) Borchert, T. V., Zeelen, J. P., Schliebs, W., Callens, M., Minke,W.,
Jaenicke, R., and Wierenga, R. K. (1995) An interface
point-mutation variant of triosephosphate isomerase is compactly
folded and monomeric at low protein concentrations. FEBS Lett.
367, 315-318.

(32) Vargo, M. A., Nguyen, L., and Colman, R. F. (2004) Subunit
interface residues of glutathione S-transferase A1-1 that are
important in the monomer-dimer equilibrium. Biochemistry 43,
3317-3335.

(33) Boulanger, R. R., Jr., and Kantrowitz, E. R. (2003) Characterization of a monomeric Escherichia coli alkaline phosphatase formed upon a single amino acid substitution. J. Biol. Chem. 278, 23497-501.
(34) Creighton, T. E. Protein structure: A practical approach. IRL Press
Oxford
(35)Righetti, P. G., and Verzola, B. (2001) Folding/unfolding/refolding of proteins: present methodologies in comparison with capillary zone electrophoresis. Electrophoresis 22, 2359-74.
(36) Beernink, P. T., and Tolan, D. R. (1996) Disruption of the aldolase A tetramer into catalytically active monomers. Proc. Natl. Acad. Sci. U S A 93, 5374-9.
(37) Cioni, P., Stroppolo, M. E., Desideri, A., and Strambini, G. B. (2001) Dynamic features of the subunit interface of Cu,Zn superoxide dismutase as probed by tryptophan phosphorescence. Arch. Biochem. Biophys. 391, 111-8.