短鏈去氫酶家族中的Comamonas testosteroni 3α-羥基固醇去氫酶/羰基還原酶 (3a-HSD/CR)與受質結合進行可逆性的氧化還原反應，催化androsterone及NAD＋形成androstanedione及NADH，其受質結合環 (T188-M213) 位於βF sheet及aG helix之間。當酵素與NAD+ 結合時，誘導受質結合環構型上的改變，促使T188與NAD+ 之nicotinamide形成氫鍵，同時導致S114與受質結合環上之P185距離增加0.19 A。為了進一步了解受質結合環之擺動性，T188和P185與NAD(H)結合時的關係及酵素結構變化，因此本文利用定點突變，設計T188A、T188S、T188W、P185A、P18G、P185L、P185W及W173F/T188W、W173F/P185W、W173F/I206W、W173F/L197W突變酵素，進一步以酵素動力學研究、圓偏振二旋圖譜、螢光光譜儀等實驗進行其研究分析。
3a-HSD/CR屬於ordered Bi Bi催化機制，在動力學參數實驗結果顯示T188及P185催化位組突變酵素對催化反應的影響。T188A之V/Et (s-1)及NAD＋的解離常數(KiNAD)比野生型分別增加8.7及5.5倍，意味著將T188取代alanine，破壞NAD＋與受質結合環上T188之氫鍵，導致增加NAD＋的解離常數。在固定5mM的NAD＋下，改變androsterone的濃度探討突變酵素活性，T188A及T188S突變酵素對kcat/Km的影響分別下降了4.7及3.9倍，在kcat則增加為3.6及7.6倍，T188S其Km增加為81；T188W與double mutant W173F/T188W對kcat/Km的影響分別下降了1.3×105及3.8×103倍，在kcat則下降為3×104及2.0×103倍。根據酵素活性降低的倍數，暗示了T188對於催化反應的重要性。proline胺基酸分子性質原本較為固定(rigid)之型態，單一突變酵素的P185A及P185G對kcat/Km的影響分別下降了15及5倍，在kcat則下降為2.7及2.1倍；P185L對kcat/Km的影響下降了667倍，在kcat則下降為24倍；P185W對kcat/Km的影響下降了1.7×103倍，在kcat則下降為619倍。由此可見，胺基酸突變後將增強催化位loop之擺動性。其double mutant W173F/P185W對kcat/Km的影響分別下降了3.8×103倍，在kcat則為1.8×103倍。W173F/I206W對kcat/Km及kcat的影響也分別增加了416.7及15.9倍，W173F/L197W在kcat/Km下降8倍及kcat的影響也增加了3倍。
CD光譜得知，3α-HSD/CR之T188突變酵素，沒有明顯的二級結構改變，但P185突變酵素造成二級結構改變，加入NADH及androsterone形成binary及ternary complex，並未進一步影響其構型。以295nm激發酵素的內生性螢光監測pH10.5的環境，突變型W173F/T188W、W173F/P185W、W173F/I206W、W173F/L197W及最高的波長由330nm分別偏移至348 nm、347 nm、349 nm及348nm，顯示出受質結合環上之T188及P185位於親水性(red shift)的環境。進一步以螢光滴定(Fluorescence titration)之方式，探討NAD+與酵素結合的解離常數Kd，T188突變型酵素增加4-6.5倍，而P185突變型酵素增加1-3.9倍，由此得知突變後的酵素確實會影響loop的結合能力及擺動功能。
綜合上述結果得知，3a-HSD/CR參與固醇類荷爾蒙代謝及催化androsterone及NAD＋進行可逆性的氧化還原反應時，Pro-185突變酵素對於受質結合環的構形及Thr-188突變酵素在輔因子結合及催化，扮演重要的角色。

The substrate binding loop (T188-M213) of 3a-hydroxysteroid Dehydrogenase /carbonyl reductase (3a-HSD/CR) of the short chain dehydrogenase/reductase superfamily Comamonas testosteroni, situated between the βF sheet and the aG helix, appearred an important variable substructure . The reversible oxidation-reduction of 3a-HSD/CR catalyzed androsterone and NAD+, formed androstanedione and NADH. A previous study showed that binding NADH with S114A mutant led to the conformational change, which could be observed by the circular dichroism measurement and fluorescence spectrum. When the enzyme was binded with NAD+, it induced the combination of quality changes on the conformation ring, prompted T188 and the NAD+ nicotinamide formation of hydrogen bonds. In addition, we observed that distance between hydroxyl group of S114 and carbonyl group of P185 located near the substrate binding loop increased by 0.19 A in the the enzyme-NAD+ binary complex. In this thesis, we characterized the roles of P185 and T188 in the substrate binding loop by site-directed mutagenesis, circular dichroism, fluorescence spectrum, and kinetic studies to probe the conformational changes. Mutants of T188A, T188S, T188W, P185A, P185G, P185L, P185W, and double mutants of W173F/T188W, W173F/P185W, W173F/I206W, and W173F/L197W 3a-HSD/CRs were generated and characterized. 3a-HSD/CR is ordered Bi Bi catalytic mechanism. Kinetic experiments showed that the T188 and P185 enzyme catalytzed group mutations on the catalytic reaction. Compared to wild-type, mutants of T188A increased the Michaelis constant Km for androsterone, inhibition dissociation constant (KiNAD) by 8.7 and 5.5 folds respectively, which meaned the T188 instead of alanine, broke NAD+ and substrate bad combination of hydrogen bonds on the ring T188 and catalytic constants kcat. T188A mutant caused the increase of the dissociation of nucleotide, thereby increasd the rate of releasing product. In the condition of of NAD+ fixed at 5mM, and change androsterone concentration of mutant enzyme activity, the impact of T188A and T188S mutant enzymes on kcat/Km decreased by 4.7 and 3.9 folds, and kcat increased by 3.6 and 7.6 folds; for T188S, Km increased to 81; for T188W and the double mutant W173F/T188W, kcat/Km decreased by 1.3 × 105 and 3.8 × 103 folds respectively, and kcat decreased by 3 × 104 and 2.0 × 103 folds respectively. The multiply decrease of enzyme activity suggested the importance of T188 in the catalytic reaction. Based on proline more rigid of the amino acid, the impact of single enzyme P185A and P185G mutations on kcat/Km decreased by 15 and 5 folds, and kcat decreased by 2.7 and 2.1 folds; for P185L, kcat/Km decreased by 667 folds and kcat decreased by 24 folds; for P185W, kcat/Km decreased by 1.7 × 103 folds while kcat decreased by 619 foldd. Thus, the amino acid mutations had enhanced the catalytic loop of the swing. For the double mutant W173F/P185W, its impact on kcat/Km, decreased by 3.8 × 103 folds and on kcat deceased by 1.8 × 103 folds. Finally, for W173F/I206W its impact on kcat/Km and kcat increased by 416.7 and 15.9 folds,while W173F/L197W its impact on kcat/Km decreased by 8 folds and kcat decreased by 3 folds.
CD spectra showed that there was no sigifinicant changes on secondary strcture of the T188 mutant enzyme of 3α-HSD/CR, while the P185 mutant enzymes caused changes in secondary structure. Adding NADH and androsterone, which formed binary and ternary complex, the conformation was not further affected. Monitoring pH10.5 environment by the fluorescence of the 295nm stimulate endogenous enzyme, the highest wavelength of the mutants W173F/T188W, W173F/P185W, W173F/I206W, W173F/L197W were offset from 330nm to 348 nm, 347 nm, 349 nm , and 349nm respectively. This showed that the substrate binding loop was located on the T188 and P185 hydrophilic (red shift) environment. Furtherly observing the dissociation constant Kd of the binding of enzyme and NAD+ by the fluorescence titration method, T188 mutant enzyme increased 4-6.5 folds, while the P185 mutant enzymes increased 1-3.9 folds. This suggested that the mutated enzyme surely affected the binding loop and swing functions.
Taking these results, in the process of 3a-HSD/CR acting on steroid hormone metabolism and the reversible redox reaction catalysis of androsterone and NAD+, P185 mutant enzymes plays a key role in the substrate binding loop conformaiton and while the T188 mutant enzymes in cofactor binding and catalysis.

目錄 I
圖目錄 III
表目錄 III
縮寫表 VII
中文摘要 VIII
英文摘要 X
壹、緒論 1
一、前言 1
(一)羥基類固醇脫氫酶(Hydroxysteroid dehydrogenases，HSDs) 1
(二)短鏈去氫酶家族(Short chain dehydrogenase/reductase superfamily，SDRs) 3
(三)Comamonas testosteroni 3α-hydroxysteroid dehydrogenase/ carbonylreductase (3α-HSD/CR) 5
(四)受質結合環(Substrate binding loop) 8
二、研究目的 11
貳、實驗材料與方法 12
一、實驗材料 12
(一)實驗藥品與試劑 12
(二)緩衝溶液及試劑配製 15
(三)儀器與器材 18
二、實驗方法 19
(一)定點突變(Site-directed Mutagenesis) 19
(二)野生型及突變型的3α-HSD/CR表達及純化 24
(三)酵素動力學基本活性測定及研究(Kinetic assay) 28
(四)圓偏振二旋圖譜分析(Circular dichroism,CD) 29
(五)螢光滴定(Fluorescence titration) 31
參、實驗結果 33
一、野生型及突變型3α-HSD/CR之定點突變、過度表達及純化 33
二、野生型及突變型3α-HSD/CR之穩定動力學研究 36
三、野生型及突變型3α-HSD/CR之圓偏振二旋圖譜(Circular dichroism,CD)分析測定 38
四、野生型及突變型3α-HSD/CR之螢光滴定 (Fluorescence titration)探討 40
肆、實驗討論 42
伍、圖表 49
參考文獻 87

參考文獻
1.Jez, J.M., et al., Comparative anatomy of the aldo-keto reductase superfamily. Biochem J, 1997. 326 ( Pt 3): p. 625-36.
2.Mellon, S.H., L.D. Griffin, and N.A. Compagnone, Biosynthesis and action of neurosteroids. Brain Res Brain Res Rev, 2001. 37(1-3): p. 3-12.
3.Heredia, V.V., R.G. Kruger, and T.M. Penning, Steroid-binding site residues dictate optimal substrate positioning in rat 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD or AKR1C9). Chem Biol Interact, 2003. 143-144: p. 393-400.
4.Seckl, J.R., 11beta-Hydroxysteroid dehydrogenase in the brain: a novel regulator of glucocorticoid action? Front Neuroendocrinol, 1997. 18(1): p. 49-99.
5.Sandeep, T.C., et al., 11Beta-hydroxysteroid dehydrogenase inhibition improves cognitive function in healthy elderly men and type 2 diabetics. Proc Natl Acad Sci U S A, 2004. 101(17): p. 6734-9.
6.Wilson, R.C., et al., A mutation in the HSD11B2 gene in a family with apparent mineralocorticoid excess. J Clin Endocrinol Metab, 1995. 80(7): p. 2263-6.
7.Suzuki, T., et al., 17Beta-hydroxysteroid dehydrogenase type 1 and type 2 in human breast carcinoma: a correlation to clinicopathological parameters. Br J Cancer, 2000. 82(3): p. 518-23.
8.Miyoshi, Y., et al., Involvement of up-regulation of 17beta-hydroxysteroid dehydrogenase type 1 in maintenance of intratumoral high estradiol levels in postmenopausal breast cancers. Int J Cancer, 2001. 94(5): p. 685-9.
9.Chetrite, G.S., et al., Dydrogesterone (Duphaston) and its 20-dihydro-derivative as selective estrogen enzyme modulators in human breast cancer cell lines. Effect on sulfatase and on 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activity. Anticancer Res, 2004. 24(3a): p. 1433-8.
10.Chetyrkin, S.V., et al., Characterization of a novel type of human microsomal 3alpha -hydroxysteroid dehydrogenase: unique tissue distribution and catalytic properties. J Biol Chem, 2001. 276(25): p. 22278-86.
11.Jornvall, H., et al., Short-chain dehydrogenases/reductases (SDR). Biochemistry, 1995. 34(18): p. 6003-13.
12.Filling, C., et al., Critical residues for structure and catalysis in short-chain dehydrogenases/reductases. J Biol Chem, 2002. 277(28): p. 25677-84.
13.Keller, B., et al., Bioinformatic identification and characterization of new members of short-chain dehydrogenase/reductase superfamily. Mol Cell Endocrinol, 2006. 248(1-2): p. 56-60.
14.Kallberg, Y., et al., Short-chain dehydrogenases/reductases (SDRs). Eur J Biochem, 2002. 269(18): p. 4409-17.
15.Kavanagh, K.L., et al., Medium- and short-chain dehydrogenase/reductase gene and protein families : the SDR superfamily: functional and structural diversity within a family of metabolic and regulatory enzymes. Cell Mol Life Sci, 2008. 65(24): p. 3895-906.
16.Grimm, C., et al., 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, 2000. 275(52): p. 41333-9.
17.Clark, D.D. and S.A. Ensign, Characterization of the 2-[(R)-2-hydroxypropylthio]ethanesulfonate dehydrogenase from Xanthobacter strain Py2: product inhibition, pH dependence of kinetic parameters, site-directed mutagenesis, rapid equilibrium inhibition, and chemical modification. Biochemistry, 2002. 41(8): p. 2727-40.
18.Hwang, C.C., et al., Mechanistic roles of Ser-114, Tyr-155, and Lys-159 in 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni. J Biol Chem, 2005. 280(5): p. 3522-8.
19.Perkins, D.I., et al., Loop 2 structure in glycine and GABA(A) receptors plays a key role in determining ethanol sensitivity. J Biol Chem, 2009. 284(40): p. 27304-14.
20.Koumanov, A., et al., The catalytic mechanism of Drosophila alcohol dehydrogenase: evidence for a proton relay modulated by the coupled ionization of the active site Lysine/Tyrosine pair and a NAD+ ribose OH switch. Proteins, 2003. 51(2): p. 289-98.
21.Chang, Y.H., L.Y. Chuang, and C.C. Hwang, Mechanism of proton transfer in the 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni. J Biol Chem, 2007. 282(47): p. 34306-14.
22.Chang, Y.H., et al., Role of S114 in the NADH-induced conformational change and catalysis of 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni. Biochim Biophys Acta, 2009. 1794(10): p. 1459-66.
23.Nakamura, S., et al., Apo- and holo-structures of 3alpha-hydroxysteroid dehydrogenase from Pseudomonas sp. B-0831. Loop-helix transition induced by coenzyme binding. J Biol Chem, 2006. 281(42): p. 31876-84.
24.Pruneda-Paz, J.L., et al., Identification of a novel steroid inducible gene associated with the beta hsd locus of Comamonas testosteroni. J Steroid Biochem Mol Biol, 2004. 88(1): p. 91-100.
25.Ladenstein, R., J.O. Winberg, and J. Benach, Medium- and short-chain dehydrogenase/reductase gene and protein families : Structure-function relationships in short-chain alcohol dehydrogenases. Cell Mol Life Sci, 2008. 65(24): p. 3918-35.
26.Nam, G.H., et al., The conserved cis-Pro39 residue plays a crucial role in the proper positioning of the catalytic base Asp38 in ketosteroid isomerase from Comamonas testosteroni. Biochem J, 2003. 375(Pt 2): p. 297-305.
27.Johnson, T.A. and T. Holyoak, Increasing the conformational entropy of the Omega-loop lid domain in phosphoenolpyruvate carboxykinase impairs catalysis and decreases catalytic fidelity. Biochemistry, 2010. 49(25): p. 5176-87.