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dc.contributor.author張晉源en_US
dc.contributor.authorChang, Chin-Yuanen_US
dc.contributor.author吳東昆en_US
dc.contributor.authorWu, Tung-Kungen_US
dc.date.accessioned2014-12-12T01:23:03Z-
dc.date.available2014-12-12T01:23:03Z-
dc.date.issued2010en_US
dc.identifier.urihttp://140.113.39.130/cdrfb3/record/nctu/#GT079328518en_US
dc.identifier.urihttp://hdl.handle.net/11536/40607-
dc.description.abstract胺醯組氨酸雙胜肽酶(PepD,EC 3.4.13.3)為胜肽酶家族M20中的一員,具有寬廣受質專一性,包括可水解肌雙胜(carnosine)及相關之加長肌雙胜(homocarnosine)以及一些三胜肽。基本上PepD可催化水解Xaa-His雙胜肽而釋放出N端的胺基酸。在此論文中,主要研究溶藻弧菌PepD的蛋白質結構、生化特性和金屬催化機轉。利用蛋白質結晶學解出PepD的晶體結構,結果顯示PepD為同源單體所組成的一個二聚體,每個單體包含一個“蓋子區域”和一個雙鋅離子依存的“催化區域”。不同於其他M20家族的二聚體,PepD二聚體結構展現一個獨特的十字構形是經由蓋子區域接觸面間的交互作用力所形成。突變分析確定幾個重要的殘基對於蛋白質結構、受質的辨認、與酵素的活性扮演著關鍵的角色。另一方面,銅離子取代PepD活性中心的雙鋅離子,結果產生兒茶酚氧化活性。我們發現”雙銅−PepD”能氧化末端帶有極性的兒茶酚衍生物,而無法氧化兒茶酚或是帶有非極性支鏈的3,5−叔丁基鄰苯二酚(DTC)。蛋白質−配體入塢結果顯示,此類帶有極性末端的兒茶酚衍生物可與”雙銅−PepD”結合。綜合言之,此研究對胺醯組氨酸雙胜肽酶的酵素結構-功能-反應機制之關係提供更進一步的了解,同時加速開發抗體−酵素導向之前導藥物治療(ADEPT)。此外,我們證實了金屬取代是造成PepD水解酵素功能酵和氧化活性之間轉換的關鍵,因此對於酵素趨異演化開啟了一個嶄新的方向。zh_TW
dc.description.abstractAminoacylhistidine dipeptidase (PepD, EC 3.4.13.3), a member of peptidase M20 family, exhibits a broad substrate specificity for unusual dipeptides carnosine (β-Ala-L-His) and homocarnosine (γ-aminobutyl-His) and a few tripeptides. Basically, PepD catalyzes the cleavage and release of N-terminal amino acid from Xaa-His dipeptide molecules. In this thesis, the PepD from Vibrio alginolyticus was physically and chemically characterized for protein 3D structure, biochemical property, and metal-catalyzed mechanism. The 3D structure was solved by X-ray crystallography, showing that PepD is a homodimer. Each monomeric subunit of the homodimer is composed of a lid domain and a catalytic domain, in which two zinc ions dwell in the active site. In distinction to other M20 family enzymes, the PepD’s dimeric structure exhibits a unique criss-cross configuration that is likely formed through interface interaction of respective lid domains. By performing mutational analysis, crucial residues were identified for maintaining PepD architecture, substrate recognition and enzymatic activity. In addition, changing the active site zinc ions with copper ions, converts PepD to catechol oxidase. The CuCu-PepD is able to oxidize catechol derivatives with a polar tail, but not catechol or 3,5-di-tert-butylcatechol (DTC) that carries non-polar side chains. This result agrees with protein-ligand docking analysis. Collectively, this study advances our overall understanding for aminoacylhistidine dipeptidase in the structure-activity relationships and facilitates future development of antibody-directed enzyme prodrug therapy (ADEPT). Most importantly, the identification of PepD functionality conversion from peptidase to oxidase has paved a new avenue for divergent enzyme evolution.en_US
dc.language.isoen_USen_US
dc.subject溶藻弧菌zh_TW
dc.subject胺醯組胺酸雙胜肽酶zh_TW
dc.subject晶體結構zh_TW
dc.subject突變分析zh_TW
dc.subject肌雙胜zh_TW
dc.subject兒茶酚zh_TW
dc.subject水解酶zh_TW
dc.subject氧化酶zh_TW
dc.subjectVibrio alginolyticusen_US
dc.subjectAminoacylhistidine dipeptidaseen_US
dc.subjectCrystal structureen_US
dc.subjectMutational analysisen_US
dc.subjectCarnosineen_US
dc.subjectCatecholen_US
dc.subjectHydrolaseen_US
dc.subjectOxidaseen_US
dc.title溶藻弧菌胺醯組胺酸雙胜肽酶之晶體結構與突變分析zh_TW
dc.titleCrystal Structure and Mutational Analysis of Aminoacylhistidine Dipeptidase (PepD) from Vibrio alginolyticusen_US
dc.typeThesisen_US
dc.contributor.department生物科技學系zh_TW
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