困難梭狀桿菌孢子皮層裂解?SleC功能與結構之研究

Taipei Medical University Institutional Repository > 醫學科技學院 > 醫學檢驗暨生物技術學系所 > 博碩士論文 > Item 987654321/63437

請使用永久網址來引用或連結此文件: http://libir.tmu.edu.tw/handle/987654321/63437

題名:	困難梭狀桿菌孢子皮層裂解?SleC功能與結構之研究 Functional and structural studies of Clostridium difficile spore cortex lytic enzyme SleC
作者:	張家菱 CHANG, CHIA-LING
貢獻者:	醫學檢驗暨生物技術學系碩士在職專班劉佳宜
關鍵詞:	困難縮狀桿菌孢子 Clostridium difficile spore
日期:	2023-06-28
上傳時間:	2024-01-22 13:23:08 (UTC+8)
摘要:	困難梭狀桿菌(Clostridium difficile)是一種生長在厭氧環境的革蘭氏陽性菌，在高溫和抗生素的惡劣條件下，具有形成孢子的獨特能力。感染困難梭狀桿菌相關的疾病稱為CDI (C. difficile infection, CDI)，標準的治療方式為抗生素使用，也因抗生素的長期使用，進而引發抗生素所造成的相關性腹瀉(Antibiotic-associated diarrhea, AAD)，困難梭狀桿菌最後會附著在人類腸道，造成偽膜性結腸炎(Pseudomembranous colitis, PMC)。困難梭狀桿菌孢子萌發(Germination)的過程需要水解皮質層(cortex)，它是由密集?聚醣所組成，皮質層對於維持孢子休眠非常重要。在皮質降解過程中，由皮質裂解水解?(cortex lytic hydrolase) SleC進行。在孢子萌發的過程中，主要的蛋白?CspB負責與具活性SleC結合，以啟動對皮質層的水解作用。缺乏sleC會導致孢子萌發過程有缺陷，無法成為具有致病力的營養細胞(Vegetative cell)。　　在這項研究中，我們首次發表了困難梭狀桿菌SleC的全長蛋白的晶體結構。此結構最高解析度可至2.24 ?，其空間群 (space group) 則是屬於C2對稱，並發現一個不對稱單元 (asymmetric unit)含有四個SleC分子，且在晶格堆疊中會形成穩定的八聚體。全長SleC蛋白質結構主要由一條β-strand與一個顯著延伸的β-turn組成propeptide，用以連接N端β-sandwich結構域（prepeptide）與C端真正具活性酵素mature SleC，它為α-helix globular結構。有趣的是，這個延伸的β-turn佔據了mature SleC活性區，可作為一種防止?聚醣(PG) 結合的調控機制。此結果顯示SleC可藉由propeptide的切割與否來調控SleC對受質辨識與受質結合專一性，突顯出其在催化功能上的重要性。這一開創性成果提供我們對SleC分子結構，並為進一步研究其功能特性和潛在應用治療提供未來方針。 C. difficile is an anaerobic, Gram-positive, rod-shaped bacterium known for its distinctive ability to form multi-layered spores under adverse conditions, including elevated temperature and exposure to antibiotics. The process of spore germination necessitates the elimination of the cortex, a dense layer of modified peptidoglycan crucial for maintaining spore dormancy. Cortex degradation is orchestrated by the SleC cortex lytic hydrolase, known for its ability to recognize the cortex-specific modification muramic-δ-lactam (MAL). In C. difficile, CspB is responsible for the processing of inactive proSleC zymogen into active SleC to initiate the enzymatic breakdown of the cortex layer. The lack of sleC leads to defective germination, which cannot become a vegetative cell. In this study, we unveil the crystal structure of the full-length SleC protein from C. difficile, marking the first characterization of its structure to date. The structure was resolved at a high resolution of 2.24 ?. PreproSleC crystallizes in the C2 space group and there are four molecules per asymmetric unit. The full-length SleC protein consists of an β-strand with a notable extending loop, referred to as the propeptide, which acts as a bridge connecting the N-terminal β-sandwich domain (prepeptide) and the C-terminal α-helix globular domain (active enzyme form). Intriguingly, this elongated β-turn occupies the groove region of the active SleC, potentially serving as a mechanism to prevent peptidoglycan (PG) binding. This suggests that the propeptide may play a role in regulating the substrate specificity or enzymatic activity of SleC by modulating access to the active site, highlighting its significance in the functional dynamics of SleC. This pioneering achievement provides a foundational understanding of the molecular architecture of SleC and paves the way for further investigations into its functional properties and potential application.
描述:	碩士指導教授:劉佳宜委員:鄭文義委員:吳昭容委員:劉佳宜
資料類型:	thesis
顯示於類別:	[醫學檢驗暨生物技術學系所] 博碩士論文

文件中的檔案:

檔案	描述	大小	格式	瀏覽次數
index.html		0Kb	HTML	39	檢視/開啟

在TMUIR中所有的資料項目都受到原著作權保護.

TAIR相關文章

資料載入中.....