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論文中文名稱:金黃色葡萄球菌糖基轉移酶 SdgB 的晶體結構 [以論文名稱查詢館藏系統]
論文英文名稱:Crystal structure of the glycosyltransferases SdgB from Staphylococcus aureus. [以論文名稱查詢館藏系統]
院校名稱:臺北科技大學
學院名稱:工程學院
系所名稱:化學工程研究所
畢業學年度:102
出版年度:103
中文姓名:張維容
英文姓名:Wei-Jung Chang
研究生學號:101738508
學位類別:碩士
語文別:中文
口試日期:2014-07-24
論文頁數:44
指導教授中文名:劉宣良
口試委員中文名:黃志宏;劉佳宜
中文關鍵詞:X 射線晶體學結構生物學絲氨酸-天門冬氨酸二肽重複序列糖基轉移酶金黃色葡萄球菌N-乙醯葡糖胺
英文關鍵詞:X-ray crystallographystructural biologySdgBserine-aspartate dipeptide-repeats (SDR)glycosyltransferasesStaphylococcus aureusN-acetylglucosamine (GlcNAc)
論文中文摘要:金黃色葡萄球菌是一種重要病原菌。在過去20年,社區型和醫院感染金黃色葡萄球菌的機率大幅增加。宿主組織感染金黃色葡萄球菌時需要一種特殊的葡萄球菌黏附蛋白,其特色為都具有絲氨酸-天門冬氨酸二肽重複序列 (SDR)。致病性葡萄球菌會表達數種 SDR 蛋白質,這些 SDR 蛋白質會受兩個糖基轉移酶SdgA及SdgB的作用而產生高度糖基化。
本研究解析出金黃色葡萄球菌糖基轉移酶SdgB 的 native型結構。其蛋白質結構和典型的糖基轉移酶相似,但額外多了一段?-sheet 結構區域。將此蛋白結構與典型糖基轉移酶的結構重疊,我們發現其中有一個SO42- 的結合位置和典型糖基轉移酶的UDP 結合位置是非常相似的。此外,我們推論在此?-sheet結構區域及周圍的一些正電性殘基,如 Arg43、Arg47、Arg73、Lys92、Arg101、Lys118、Arg120、Arg132、 Lys134、Arg137 和Arg-150等,是與SDR 結合的重要殘基。總結來說,該結構提供了一個研究SdgB糖基化機制的機會。
論文英文摘要:Staphylococcus aureus is an important pathogen. Both community-associated and hospital-acquired infections with S. aureus have increased in the past 20 years. Infection of host tissues by S. aureus requires a specific group of staphylococcal adhesive proteins with serine-asparate dipeptide repeats (SDRs). Pathogenic staphylococci express several SDR proteins that are heavily glycosylated by two glycosyltransferases, SdgA and SdgB. In this study, the structure of SdgB from S. aureus was determined in apo. The structure of S. aureus SdgB is similar to that of typical glycosyltransferases, despite having an extra ?-sheet domain. Superimposition of the S. aureus SdgB structure with typical glycosyltransferase structures, we found that one of the SO42- binding sites is very similar to that of UDP binding site in typical glycosyltransferases. In addition, we propose some positive residues, such as Arg43, Arg47, Arg73, Lys92, Arg101, Lys118, Arg120, Arg132, Lys134, Arg137 and Arg150 located in the ?-sheet domain serve as important residues responsible for binding with SDR. In summary, the structure of S. aureus SdgB provides an opportunity to investigate the mechanism of SdgB-mediated glycosylation.
論文目次:中文摘要...................i
英文摘要...................ii
誌謝...................iii
目錄...................iv
表目錄...................vi
圖目錄...................vii
第一章 前言...................1
1.1細菌細胞壁...................1
1.2糖基轉移酶(Glycosyltransferases, GT...................2
1.3 Staphylococcus aureus...................5
1.4 Staphylococcus aureus SdgB...................6
第二章 材料與方法...................9
2.1材料...................9
2.2 SdgB cloning...................9
2.2.1 從Staphylococcus aureus的genomic DNA擴大SdgB..........9
2.2.2 將S. aureus SdgB接合至pGEM-T vector...................9
2.2.3 pGEM-T SdgB藍白篩選和定序...................10
2.2.4 將S. aureus SdgB接合至pET-28 vector..................11
2.3 蛋白表現及純化...................12
2.3.1 S. aureus SdgB 蛋白表現...................12
2.3.2 S. aureus SdgB 蛋白純化...................12
2.3.3 Seleno-methionine labelled S. aureus SdgB 蛋白表現..13
2.3.4 Seleno-methionine labelled S. aureus SdgB 蛋白純化..13
2.4結晶實驗...................13
2.5 X-ray數據收集、處理...................15
2.6結構確定及Refinement...................15
第三章 結果...................16
3.1 SdgB cloning...................16
3.2 S. aureus SdgB 蛋白表現及純化...................20
3.3 S. aureus SdgB and SeMet S. aureus SdgB crystal.....21
3.4 S. aureus SdgB 整體結構...................23
3.5 S. aureus SdgB 結構類似物(structure homolog)...........30
3.6 S. aureus SdgB 的β – sheet區域...................30
3.7 S. aureus SdgB 的活性區...................30
第四章 討論...................40
第五章 結論與建議...................41
參考文獻...................43
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