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  • 學位論文

疫病菌多聚半乳糖醛酸酶(pppg1)之分子選殖與功能性分析

Molecular cloning and functional analysis of pppg1 encoding endo-polygalacturonase in Phytophthora parasitica

指導教授 : 劉瑞芬

摘要


疫病菌pppg1基因之蛋白產物為一個可表現多聚半乳糖醛酸酶酵素活性之醣蛋白,胺基酸序列分析顯示其具有11個可能之醣基化位置,但對於這些位置是否真有醣基化反應發生及醣基與酵素活性之間的關連性目前仍不清楚。為了解pppg1醣基化反應與其酵素活性之間的相關性,本實驗利用定點突變的方式針對醣基化位置進行一系列單點及多點突變,並以酵母菌(Pichia pastoris)系統表現重組蛋白,以便進行酵素活性及生化特性分析。分析結果顯示,這11個可能之醣基化位置確實都有醣基化反應發生。進一步分析這些突變蛋白的酵素活性,發現每一個醣基化位置都對酵素活性具有重要作用,特別是在N8位置的醣基化反應對酵素活性的影響最大。實驗也發現,N1、N3、N4、N10與N11等位置的醣基化反應對於pppg1重組蛋白在高溫逆境下維持穩定性非常重要。此外,醣基化反應也與pppg1蛋白的分泌有密切關係。另一方面,為探討pppg1蛋白質N端的醣基與蛋白質折疊之間的關係,一個未含醣基化位置之多聚半乳糖醛酸酶(PG3b)基因的N端與PPPG1的N端進行交換以得到重組蛋白PG3b1a,酵素活性分析結果發現,雖然重組蛋白PG3b1a的N端缺乏醣基,卻還是具有很強的酵素活性,顯見pppg1蛋白質原本N端之醣基化反應對於蛋白質結構的完整性很重要。然而,重組蛋白PG3b1a對高溫仍較為敏感,顯示在高溫情況,醣基對於蛋白的保護功能是無法取代的。同時,我也針對多聚半乳糖醛酸酶的保守性胺基酸進行突變分析,實驗結果發現Asp209、 Asp230、Asp231、H252與K290與酵素活性有重要關係。

並列摘要


The deduced amino acid sequence of pppg1, which encodes a polygalacturonase in the oomycete plant pathogen Phytophthora parasitica, contains eleven putative N-glycosylation sites. To understand the significance of N-glycosylation on the enzymatic activity of PPPG1, site-directed mutagenesis was employed to generate a serial of single and multiple mutations on the N-glycosylation sites. Recombinant mutant proteins were expressed using a yeast (Pichia pastoris) protein expression system and their biochemical characteristics were analyzed. The results indicated that mutations in the N-glycosylation sites resulted in a significant loss of the endoPG activity compared to that of the wild-type protein, especially mutation at N8, suggesting the importance of N-glycosylation on the structure and thereby the enzymatic function of the PPPG1. Besides, mutation in positions N1, N3, N4, N10, and N11 caused a reduction in thermostability of the recombinant proteins. It is thus suggested that N-glycosylations on these amino acid residues might be involved in preventing the protein from the deleterious effect caused by heat. N-glycosylation is also important for the secretion of PPPG1. Furthermore, it was demonstrated that the replacement of the N-terminus of PPPG1 with that of PG3b, another gene encoding endopolygalacturonase in P. parasitica which contains no N-glycosylation site, resulted in restoration of activity to N1-4 mutated PPPG1, indicating that N-glycosylation in the first four N-glycosylation sites of pppg1 might play a major role in the formation of parallel β-helix. Nevertheless, the chimeric protein exhibited a reduction in thermostability, implicating the essential function of N-glycosylation in increasing the stability of the enzyme against thermal unfolding. In the meanwhile, mutations were generated in six amino acid residues which are highly conserved among endoPGs. Analysis of the biochemical properties of these mutants indicated that residues Asp209, Asp230, Asp231, H252, and K290 play very important roles in pectin hydrolysis.

並列關鍵字

Phytophthora parasitica polygalacturonase PG

參考文獻


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張怡萱(2013)。疫病菌外泌蛋白OPEL在植物反應所扮演的角色〔碩士論文,國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342%2fNTU.2013.10966

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