Xanthomonas campestris pv. campestris未知功能蛋白XC847的晶體結構及功能分析;Structural determination and functional analysis of unknown function protein XC847 in Xanthomonas campestris pv. campestris

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題名:	Xanthomonas campestris pv. campestris未知功能蛋白XC847的晶體結構及功能分析;Structural determination and functional analysis of unknown function protein XC847 in Xanthomonas campestris pv. campestris
作者:	吳彥宥;yen-yu wu
貢獻者:	生命科學研究所
關鍵詞:	結構基因體計畫;Xanthomonase campestris pv. campestris;ε186;Pop2;NMR;X-ray;oligoribonuclease;DEDDh family;ISG20
日期:	2005-06-27
上傳時間:	2009-09-22 10:19:07 (UTC+8)
出版者:	國立中央大學圖書館
摘要:	本論文研究的菌株為Xanthomonase campestris pv. campestris。其含有特殊調節系統（Clp，cAMP receptor protein-like protein），轉錄單位多以單基因方式，與一般細菌不同。又可分泌多醣體(Xanthan gum)，具工業價值。但它亦會引起十字花科植物的黑腐病並造成農業巨大的損失。藉由基因體解碼下，此一病原菌被預測的基因約有4100個。因此本論文用了五種不同的載體，並利用E.coli BL21宿主來大量的表逹及篩選可溶性蛋白供NMR及X-ray做分析。此二種解析結構的方法有不同的原理及限制，不過此二種方法可互補。本研究中一共挑選了15個基因，其中4個(XC4107 、XC3183 、XC6232、 XC6774)在PCR階段、7個(XC2797、XC4109、XC6773、XC6835、XC3953、XC4027、XC5047)在表現階段、2個（XC5979、X4187）在光譜測定階段、1個(XC1014)在晶體篩選及1個(XC847)已得到結構。XC847由蛋白質序列比對下，初步預測其功能為oligoribonuclease，被歸為exoribonuclease六個種類中的DEDDh家族中的一員，其含有4個高保留胺基酸DEDDh在3個功能區中。另外比對XC847和DEDDh家族的三個（ISG20、Pop2及ε186）RNase或DNase的結構，結果顯示此XC847和此3個酵素有相同的活性區域。由表面電荷分析，不同exribonuclease在活性中心帶有相同的負電荷分布，而XC847在活性中心也有相同的分布。故由以上結構及序列比對結果說明XC847可能為一個oligoribonuclease。 In this thesis, we have choosen Xanthomonas campestris pv. campestris as our target genome. It is a gram-negative bacterium that is phytopathogenic to cruciferous plants and causes worldwide agricultural loss. However, it also produces exopolysaccharide (xanthan gum) that is of great industrial importance. About 4100 genes are predicted in this genome. Five different vectors are used to construct clones and over-express proteins in the E.coli host to produce enough soluble proteins for X-ray and NMR analysis. Until now, 15 target genes were studied. 4 genes (XC4107 、XC3183 、XC6232、 XC6774) are in the PCR stage, 7 genes (XC2797、XC4109、XC6773、XC6835、XC3953、XC4027、XC5047) are in the overexpression stage, 2 are being analyzed by NMR, 1 being screened for crystallization , and one (XC847) of which the structure has been successfully determined. From sequence alignment, XC847 is predicted as an oligoribonuclease that belongs to the DEDDh family. DEDDh family is one of the six members in the 3' to 5' exonuclease superfamily, and is defined by four conserved acidic residues distributed among three separated sequence motifs. Proteins in this family can hydrolyze both DNA and RNA substrates. From the determined 3D structure, XC847 was found to have conserved residues and active site geometry similar to those in the DEDDh family, ISG20、Pop2, andε186. Moreover, the electrostatic surfaces of these 3 exoribonucleses show similar negative charged profile in their active site regions.From sequence alignment and structural comparison, XC847 is identified as an oligoribonuclease.
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