- 學位論文
3α-羥基固醇去氫酶/羰基還原酶中组胺酸119及酪胺酸202參與蛋白質穩定性、受質特異性及催化功能之探討
Studies on the function of H119 and Y202 in 3α-hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) involving in the protein stability, substrate specificity and catalysis
摘要
3α-Hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR)屬於短鏈去氫酶家族(short-chain dehydrogenase/reductase (SDR) superfamily)的一員，SDR superfamily蛋白質間在結構上有高度保留的能與cofactor結合的GxxxGxG motif及catalytic tetrad (Tyr, Lys, Ser, Asn)，然而在C-端的受質結合環(substrate binding loop)卻有高度的變異性。Comamonas testosteroni 3α-HSD/CR C-端的受質結合環(substrate binding loop)由於擺動性高，因此儘管有apo enzyme及與NAD+ 結合形成binary complex的結晶，但受質結合環的結晶結構及ternary complex未解出，透過同源性分子模擬，推測H119與位於受質結合環上的Y202與受質androsterone的C-17 carbonyl group相對距離相近。為了了解H119與Y202在3α-HSD/CR蛋白質穩定性、受質特異性及催化功能中扮演的角色，本篇研究利用定點突變，設計Y202A、H119A、H119N、H119A/Y202A突變酵素，進一步以穩定動力學研究、圓偏振二旋圖譜分析、內源性螢光研究、螢光滴定、外源性ANS binding螢光研究、尿素變性、差示掃描螢光法(DSF)進行研究分析，探討H119及Y202兩位點對3α-HSD/CR催化反應、受質特異性及整體蛋白質穩定性的貢獻。在不同受質之酵素動力學研究顯示WT 以cyclohexanol為受質的kcat/Km與以androsterone為受質相比，下降9.85×105倍，突變型H119A、H119N、Y202A、H119A/Y202A分別下降7.05×105、7.02×105、4.85×104、8.46×103 倍。以androsterone為受質當降低pH環境的酵素動力學研究顯示，在pH 6.0及pH 6.5 環境WT的kcat/Km及Km低於H119A及H119N，顯示質子化形式的histidine會降低受質特異性及酵素活性。圓偏振二旋圖譜分析結果顯示突變及配體結合不會明顯地影響整體蛋白質二級結構。內源性螢光研究及外源性ANS binding螢光研究分別說明H119及Y202突變不會影響蛋白質W173及疏水性區域的大幅度暴露，兩實驗的最高螢光強度波長分別約為330 nm及475 nm。NADH螢光滴定實驗結果顯示H119A及H119/Y202A皆會增加1.2倍NADH解離常數。尿素變性實驗之野生型及突變型3α-HSD/CR的[UREA]0.5 相似，H119N 突變型3α-HSD/CR的ΔGH2O為9.66 kcal mol-1且m為4.01 kcal mol-1 M-1，與其他組相比數值最高。在pH 4.5到pH 10.5環境之差示掃描螢光實驗(DSF)，野生型及突變型3α-HSD/CR皆在酸鹼環境Tm較中性環境低， Y202A突變型3α-HSD/CR與WT的Tm變化趨勢相似，而H119A及H119A/Y202A在鹼性環境明顯降低Tm，H119N在鹼性環境Tm的降低程度最大。pH 7.5環境之WT、H119A、H119N、Y202A、H119A/Y202A的Tm分別51.6°C、50.2°C、48.8°C、51.3°C、50.2°C。pH 9.0環境下WT 的Tm為50.9°C，分別加入NAD+或NADH的Tm為52.0°C、51.7°C，同時加入NAD+和androsterone sulphate的Tm為55.0°C，顯示配體加入會增加野生型3α-HSD/CR的熱穩定性。Protein stability curve顯示H119及Y202突變會增加3α-HSD/CR的Ts數值，WT在Ts溫度下的ΔGu較突變型3α-HSD/CR高，為7.91 kcal mol-1，在25°C下的ΔGu WT有最高數值(7.91 kcal mol-1)，而H119N為6.66 kcal mol-1數值最低。綜合本篇研究實驗，得知H119與Y202以協同作用影響3α-HSD/CR的催化效率，當受質失去固醇類BCD-ring兩位點對酵素協同作用的影響不及androsterone為受質，而H119的質子化會降低受質特異性及酵素活性。H119A突變會降低3α-HSD/CR與NADH的結合能力，二級結構則是無明顯改變。對照Molecular simulation分析，可得知H119確實會與androsterone D-ring有相互作用，當失去此作用則可能會使3α-HSD/CR的受質結合環無法被誘導產生構型改變，進而影響酵素催化能力以及造成熱穩定性不佳。
並列摘要
3α-Hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) is a member of short-chain dehydrogenase/reductase (SDR) superfamily. The structures of this superfamily are highly conserved with similar cofactor binding site GxxxGxG motif and catalytic tetrad : Tyr, Lys, Ser, Asn. The variability within SDRs is a highly variable substrate binding loop located in the C-terminal. The substrate binding loop is unresolved in crystal structure of Comamonas testosteroni 3α-HSD/CR. Based on the homology modeling of ternary complex of 3α-HSD/CR-NAD+-androsterone, histidine 119 is likely to interact with the C-17 carbonyl group of androsterone, although it is located at the opposite side of substrate binding loop. In this research, we characterize the role of H119 and Y202 in catalysis and stability of 3α-HSD/CR by site-directed mutagenesis, steady-sate kinetic, fluorescence titration, circular dichroism, and differential scanning fluorimetry (DSF). The mutants of H119A, H119N, Y202A, and H119A/Y202A were cloned, overexpressed, and purified to homogeneity. When cyclohexanol was used as substrate, the kcat/Km of WT, H119A, H119N, Y202A and H119A/Y202A mutant enzymes were decrease by 9.85×105, 7.05×105, 7.02×105, 4.85×104 and 8.46×103-folds, respectively, as compare to that of androsterone. The steady-state kinetics show that the kcat/Km of WT is lower than that of H119A and H119N at pH 6.0 and pH 6.5, indicating that the protonation state of histidine can decrease the substrate specificity, kcat/Km, and kcat. Circular dichroism results indicate that mutation on H119 in the absence and presence of ligand does not change the secondary structure of 3α-HSD/CR apparently. Intrinsic fluorescence and ANS binding assay show that mutation on H119 do not expose W173 and hydrophobic region dramatically. The λmax for both experiments were at 330 nm and 475 nm, respectively. The binding of NADH with wild-type and mutant enzymes decreases the fluorescence at 330 nm and is concomitant with energy transfer to the bound NADH when tryptophan residue is excited at 295 nm. The dissociation constant Kd of NADH obtained by fluorescence titration increases both 1.2-folds for H119A and H119A/Y202A mutants at pH 7.5. The values of [UREA]0.5 were similar for wild-type and mutant 3α-HSD/CR. ΔGH2O and m of H119N which were 9.66 kcal mol-1 and 4.01 kcal mol-1 M-1,respecyively, which were the highest among wild-type and other mutant 3α-HSD/CR. To further study the role of H119 in conformational stability, we determine the melting temperature (Tm) for apo enzyme, binary and ternary complexes by DSF. The Tm value was 51.6°C for wild-type enzyme and decreased to 50.2°C, 48.8°C, 51.3°C and 50.2°C for H119A, H119N, Y202A and H119A/Y202A at pH 7.5, respectively. The ternary complex of WT-NAD+-androsterone sulphate significantly increased the Tm value to 55.0°C for wild-type enzyme but moderately increases to 52.0°C and 51.7°C for binary complex of WT-NAD+ and WT-NADH at pH 9.0. The Tm values of wild type, H119A and H119N mutant enzymes were varied depending on pH-profile. In alkaline and acidic pH, Tm values were lower than neutral pH. Y202A have similar pH-profile pattern as WT. However, H119A and H119A/Y202A decreased the Tm in alkaline pH. Moreover, the Tm values of H119N diminish severely. Protein stability curve demonstrates that mutation on H119 and Y202 increase TS. Compared to mutant 3α-HSD/CR, WT with the highest values of ΔGu,Ts and ΔGu,25℃ (7.91 kcal mol-1) suggested the best stability. On the other hand, H119N with the lowest value of ΔGu,25℃ (6.66 kcal mol-1) showed the lowest thermal stability. In summary, mutation on H119 affects catalytic efficiency of 3α-HSD/CR and the dissociation constant with NADH. Moreover, the catalytic efficiency of H119 and Y202 mutant 3α-HSD/CRs showed the synergic effect. The protein stability changes were observed for apo enzyme, binary and ternary complexes through thermal unfolding in the presence and absence of ligands or in the different pH. H119 may interact with D-ring of androsterone. When loss this interaction, the substrate binding loop cannot be induced conformational change for better catalysis and protein thermal stability.
參考文獻
延伸閱讀
- 徐嫺樺（2003）。3a羥基類固醇脫氫酶絲胺酸114在催化反應機構及酸鹼催化作用中所扮演功能之探討〔碩士論文，高雄醫學大學〕。華藝線上圖書館。https://www.airitilibrary.com/Article/Detail?DocID=U0011-0707200816054147
- Huang, H. M., & Chen, W. Y. (2002). 甲基丙烯酸三團聯共聚物螯合改質及其於蛋白質親和性共沈澱之應用. Journal of the Chinese Institute of Chemical Engineers, 33(4), 397-405. https://doi.org/10.6967/JCICE.200207.0397
- Chen, J. J. (2020). 胍丁胺乙醯基轉移酶突變蛋白的單元體及二元體/三元體晶體結構研究以探討酵素活性之關鍵殘基 [master's thesis, National Tsing Hua University]. Airiti Library. https://www.airitilibrary.com/Article/Detail?DocID=U0016-2502202114214245
- 曾敬雅（2009）。酪氨酸磷酸化在誘發型一氧化氮合成酶蛋白質穩定度、二聚體形成、蛋白質交互作用中之角色探討〔碩士論文，中山醫學大學〕。華藝線上圖書館。https://doi.org/10.6834/CSMU.2009.00017
- 陸永軒（2019）。Study on the Development of Drugs Treating Neurological Disorders: Synthesis of the Ganglioside Hp-s1’s Analogues with Heptadecacarbon Isophtosphingosine-Phytoceramide Moiety Containing Different Length of Carbon Chain Fatty Acids〔碩士論文，中原大學〕。華藝線上圖書館。https://doi.org/10.6840/cycu201900831