Deinococcus radiodurans的同源蛋白質CN hydrolase-acyltransferase的晶體結構及酵素分析

Crystallographic and enzymatic studies of a CN hydrolase-acyltransferase homologue from Deinococcus radiodurans

許登傑(Teng-Chieh Hsu)

指導教授 ： 王雯靜

國立清華大學/生命科學院/分子與細胞生物研究所/碩士(2006年)

摘要

Nitrilase superfamily的成員為一種 thiol 的酵素，在植物、動物、真菌特定的原核生物中的自然產物的生合成和以及轉譯後的修飾，主要催化水解和縮合CN鍵。Nitrilase superfamily被分類為：有九個已經知道或是推論對於 nitrile 或 amide 的水解反應或是 amide 的縮合反應。在耐輻射奇異球菌中nitrilase superfamily的成員，CN hydrolase-acyltransferase homologes　(基因編號為DR2433)，序列與其nitrilase/cyanide hydratase 或 amidase 有高相同性。與nitrilase superfamily的序列比較e-value及z-core，推測屬於nitrilase superfamily的第十二類。本文研究結構功能相關性及其生化活性測試。Native 和SeMet的晶體都同一個條件0.1M MES pH6.0，0 2M NaCl， 0.05M KH2PO4/NaH2PO4且在 20℃中培養而成。空間群定為 P212121，而晶格參數為 a＝ 56.10 Å， b＝ 110.37 Å， c＝ 113.05 Å。晶體結構利用 multiwavelength anomalous dispersion (MAD) method，其解析度可達到 2.1Å 下決定，最小不對稱單元為一雙元體（分子量約為 64kDa），且晶體水含量為53%。每一個單體具有 α-β-β-α 三明治的構形。在表面helix作用下，雙體成為八層的 α-β-β-α-α-β-β-α 結構。在溶劑易接近的催化中心視為有Glu49、Lys127 和Cys161。進行不同種類的受質酵素分析，在反應溫度37℃下，目前只測得對於formamide具有活性，以 formamide為受質，測得最適溫度為65℃。

關鍵字

結晶 ； 酵素分析 ； 同源蛋白質

並列摘要

Members of the nitrilase superfamily are thiol enzymes that catalyze carbon-nitrogen bond hydrolysis and condensation reactions in natural products biosynthesis and protein modification in plants, animals, fungi and certain prokaryotes. Thirteen branches are defined in this superfamily: nine of which have known or deduced specificity for specific nitrile- or amide-hydrolysis or amide condensation reactions.In Deinococcus radiodurans, the CN hydrolase-acyltransferase homologue that shares sequence homology with high homology with nitrilase/ cyanide hydratase or amidas, which may belong to 12th of branch in the nitrilase superfamily protein. This thesis aims to study the structure-function relationship and biochemistry analysis of the homologue protein .Native and SeMet Crystals grew in the same conditions using 0.1M MES pH6.0, 2M NaCl, 0.05M KH2PO4/NaH2PO4 at 20℃. Crystal belongs to the orthorhombic space group P212121, with unit-cell parameters a＝ 56.10 Å ,b＝ 110.37 Å ,c＝ 113.05 Å. The crystal structure has been determined to 2.1Å resolution by the multiwavelength anomalous dispersion (MAD) method.There is a dimer (Molecular weight approximately 64 kDa) in the asymmetric unit, with a solvent content of 53%. The monomer shows a characteristic nitrilase superfamily that consists of a α-β-β-α sandwich fold . The dimer forms an eight-layer α-β-β-α-α-β-β-α structure by contants from surface helix. The catalytic center in solvent-accessible cleft could be identified Glu49 , Lys127 and Cys161. We anaylsis the enzyme activity of some kinds of nitrile and amide . The enzyme only has a acitity with formamide in reaction tempature 37℃.. Its the optimal tempature is 65℃ for formamide..

並列關鍵字

Deinococcus radiodurans ； nitrilase superfamily ； cn hydrolase ； acyltransferase

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