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標題: | 唾液酸合成酶:結構分析與鑑定參與催化反應的胺基酸殘基 Sialic Acid Synthase from Escherichia coli and Streptococcus agalactiae: Structural Characterization and Identification of Essential Catalytic Residues |
作者: | Tzann-Shun Hwang 黃贊勳 |
指導教授: | 林俊宏 |
關鍵字: | 唾液酸合成酶, Sialic Acid Synthase,Escherichia coli,Streptococcus agalactiae, |
出版年 : | 2005 |
學位: | 博士 |
摘要: | 大腸桿菌K1可導致新生兒腦膜炎,其菌體表面的莢膜含有alpha2,8鍵結的聚唾液酸,是感染寄主細胞的致病因子。無乳糖鏈球菌的莢膜含有alpha2,3鍵結的唾液酸,能抑制補體C3b的作用及防止巨噬細胞的吞噬,造成新生兒敗血症或腦膜炎。此兩菌株負責唾液酸合成的基因都是位於kps 基因叢的neuB,其基因產物為唾液酸合成酶 (EC 4.1.3.19, NeuB),可催化ManNAc與PEP的縮合反應,生成唾液酸。此酵素的產物與病源菌的致病性相關,因此對酵素活性區的探討,有助於藥物的開發,以及此酵素在唾液酸合成的應用。
本研究將大腸桿菌K1的唾液酸合成酶 (EcNeuB) 和無乳糖鏈球菌的唾液酸合成酶 (SaNeuB) 從基因體DNA中選殖出來,建立酵素的最適反應條件,包括最適酸鹼度、溫度、穩定度和金屬離子的需求等特性,並且進行生化特性的探討。EcNeuB在純化時,發現在Lys280處會被蛋白質水解酶切割,使原本約40 kDa的蛋白質,斷裂成33 kDa及7 kDa兩個片段,造成酵素失去活性。以MALDI-TOF-MS和化學鏈結法進行分析,顯示斷裂後的蛋白質會由原本的四聚體變成三聚體,以圓二極光譜儀分析40 kDa及33 kDa的二級結構,發現斷裂會造成33 kDa蛋白質alpha-helix比例的減少及beta-sheet比例的增加。SaNeuB以化學鏈結法分析也呈現四聚體的結構。以nano-spray ESI-MS及電子顯微鏡對EcNeuB和SaNeuB的四級結構做進一步的探討,發現這兩個酵素是以二聚體二聚體的交互作用形成的四聚體結構。 利用專一性化學修飾法及定點突變法,針對活性區內可能參與金屬與PEP結合的半胱胺酸殘基(Cys)進行探討。Cys的修飾試劑作用可完全抑制EcNeuB和SaNeuB的活性,並且活性會隨試劑作用的時間及濃度增加而降低。DTNB滴定分析顯示EcNeuB和SaNeuB皆沒有雙硫鍵的存在,證明修飾試劑造成酵素失去活性,是因為酵素失去重要的Cys,而與雙硫鍵的破壞無關。受質保護實驗顯示PEP+Mn2+可保護酵素不被IAA或BrPy作用,使酵素維持一定的活性,證實重要的Cys是位於活性區中,而且與PEP及Mn2+的結合有關。定點突變法、酵素活性及動力學分析顯示EcNeuB的突變株C12A和C176A與SaNeuB的突變株C10A和C169A的活性降低,且對受質PEP+Mn2+的親合力也明顯降低,但在所有定點突變株與野生型的圓二極光譜分析上並沒有顯著的差異,顯示EcNeuB的Cys12和Cys176和SaNeuB的Cys10和Cys169是與受質PEP及Mn2+結合有關。以BrPy標誌的SaNeuB進行質譜分析,結果發現被標示的Cys出現在含有Cys10和Cys169的胜肽片段上,再次證實Cys10和Cys169確實位於活性區內。以腦膜炎雙球菌唾液酸合成酶的晶體結構進行SaNeuB結構的分子模擬,以瞭解Cys10,Cys169在空間中的位置及可能扮演的功能與角色。模擬的SaNeuB結構顯示Cys169會和受質PEP的磷酸基產生交互作用,與受質PEP的結合有關,Cys10則被發現會和Gln37產生交互作用,間接影響酵素對受質 PEP的結合能力。 The capsular polysaccharide of Escherichia coli K1 containing alpha2,8-linked polysialic acid has been recognized as an important virulent determinant to cause invasive infections such as neonatal meningitis and septicemia. The capsular polysaccharide of Streptococcus agalactiae (Group B Streptococci, GBS) containing alpha2,3-sialylated oligosaccharide is also a critical virulence factor for causing neonatal septicemia, pyogenic meningitis and pneumonia. The sialic acid presented in GBS capsule has been found enhancing resistance to phagocytosis by inhibiting the alternative pathway of complement cascade to evade host defenses. In E. coli and S. agalactiae, the gene responsible for the biosynthesis of sialic acid is the neuB gene in kps gene cluster. Sialic acid synthase (NeuB), encoded by neuB gene, can catalyze the condensing reaction of N-acetylmannosamine (ManNAc) and phosphoenolpyruvate (PEP) to form N-acetylneuraminic acid (NeuAc). The product of NeuB is relative to the infection of pathogens; therefore, the investigation on the active site of NeuB is a good approach for drug design and enzyme’s application. In this study, neuB genes from E. coli and S. agalactiae were cloned from genomic DNA and over-expressed as EcNeuB and SaNeuB, respectively. Optimal conditions for enzyme reaction, including pH, temperature, stability and metal requirement, were established. Characterization of EcNeuB and SaNeuB was also conducted. In the preparation of EcNeuB, a specific cleavage by endogenous protease(s) was found at Lys280 of sialic acid synthase (40 kDa). The cleavage results in the formation of two inactive fragments of 33 kDa and 7 kDa. The CD, MALDI-TOF-MS and chemical cross-linking studies demonstrated that the fragmentation is associated with a significant change of the enzyme from a tetrameric to trimeric form. Further studies by nano-spray ESI-MS and electron microscopy demonstrated NeuB existed in a tetrameric form by dimer-dimer interaction. Sulfhydryl-modifying reagents were able to completely inactivate the enzyme activity. The iodoacetic acid (IAA) inactivation of EcNeuB and SaNeuB was in a time- and dose-dependent manner, which revealed that Cys was important for enzyme activity. Study of the sulfhydryl group by 5,5-dithiobis-2-nitrobenzoate (DTNB) titration showed no disulfide bond in both EcNeuB and SaNeuB, suggesting the activity loss was caused by the modification of Cys residue. Site-directed mutagenesis, enzyme assay and kinetic analysis showed that C12A and C176A of EcNeuB and C10A and C169A of SaNeuB were important for enzyme activity. The substrate protection experiments indicated that aforementioned Cys residues were located in the active site and involved in the binding of PEP and Mn2+, since the substrate binding could prevent NeuB from the inactivation of IAA and bromopyruvate. Further studies on substrate protection of SaNeuB mutants and molecular modeling of SaNeuB showed that Cys169 was a residue involved in the binding of PEP and Mn2+. On the other hand, Cys10 was proposed to interact with Gln37 that is essential for the binding of PEP. Chemical modification and site-directed mutagenesis showed Arg301 and Arg277 of SaNeuB were essential in the substrate binding. Molecular modeling of SaNeuB showed that Arg301 and Arg277 were involved in the binding of ManNAc and PEP, respectively. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/24262 |
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顯示於系所單位: | 生化科學研究所 |
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