Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23656
標題: 高鹽甲烷太古生物腺苷高半胱胺酸水解酵素基因與預測結構之分析
Gene and putative protein structure of S-adenosylhomocysteine hydrolase from Methanohalophilus portucalensis FDF1T
作者: 葉俊麟
Yeh, Chun-Lin
關鍵字: betaine;腺苷高半胱胺酸水解酵素;S-adenosylhomocysteine hydrolase;S-adenosylhomocysteine metabolism;腺苷高半胱胺酸代謝
出版社: 生命科學系所
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摘要: 
S-adenosylmethionine (SAM)依賴型甲基轉移反應會釋出反應代謝產物S-adenosylhomocysteine (SAH),SAH也做為此類甲基轉移酶的競爭抑制物,而SAH水解酶(SAHH)則藉由分解SAH產生adenosine和homocysteine來維持甲基化反應的進行。嗜鹽甲烷太古生物Methanohalophilus portucalensis FDF1T可將glycine經由三次SAM依賴型甲基化反應生成並累積甜菜鹼作為滲透壓保護物質,因此細胞內應具有高效率的SAH水解活性以利於相容質甜菜鹼的持續生合成。以M. portucalensis FDF1T細胞粗萃取液測試SAH水解反應證實其胞內具有SAH水解活性。經由M. portucalensis FDF1T metagenomic sequence資料與比較基因體分析胺基酸發現Methanohalophilus有兩套不同型的sahh基因,分別位於glycine betaine生合成基因組的上下游,且由序列分析得知催化活性相關的胺基酸和功能區塊與其他物種的SAH水解酶有高度的相似性,推測兩套SAHH皆具有水解SAH的能力。序列比對與結構預測分析發現MpSAHH1具有多數真核生物與細菌SAHH特有的insertion sequence與C-terminal domain,MpSAHH2則與太古生物SAHH同樣缺少了這些序列。進一步演化歸群分析將MpSAHH1與阿拉伯芥和細菌SAHH歸群在一起,MpSAHH2則和其它甲烷太古生物的SAHH歸群在一起,顯示MpSAHH1可能藉由水平傳播的方式獲自其他物種。將已獲得全長的Mpsahh1基因構築在表現載體pET28a+,以E. coli strain BL21 (DE3)RIL大量表現蛋白並以鎳離子親合性層析純化蛋白並偵測活性,結果無法測得SAH水解活性。由於SAHH需形成四聚體結構才具有功能,因此需先獲得四聚體的MpSAHH1再分析SAH水解活性。本篇研究的結果顯示嗜鹽甲烷太古生物M. portucalensis FDF1T具有兩套SAHH蛋白參與胞內甲基化反應。

S-adenosylhomocysteine hydrolase (SAHH) catalyzes the hydrolysis of SAH, which is an inhibitor of S-adenosylmethionine (SAM)-dependent methyltransferase, to form adenosine and L-homocysteine. The halophilic methanogen Methanohalophilus portucalensis FDF1T could procee de novo synthesis of glycine betaine as osmolyte via three steps SAM-dependent methylation. This infers that an efficient regulation of SAHH must exist in this organism in order to accumulate and maintain the glycine betaine level under hypersaline environment. SAH hydrolytic activity was detected in the crude extract of M. portucalensis FDF1T. Metagenomic and comparative genomics analysis of genus Methanohalophilus revealed there are two sahh genes located up and down stream of glycine betaine synthesizing gene cluster, respectively. Amino acid sequence analysis and homology modeling structure of these two MpSAHHs exhibit conserved adenosine binding sites and cofactor NAD+ binding motifs with other species, suggesting both possess SAH hydrolyzed activities. Interestingly, sequence alignment and putative structure revealed MpSAHH1 displayed an insertion sequence and C-terminal domain which were conserved among most of eukaryotic and bacterial SAHHs, while MpSAHH2, as archaeal SAHHs, do not have insertion sequence and C-terminal domain. Phylogenetic analysis revealed MpSAHH1 was clustered with SAHHs from Arabidopsis thaliana and bacteria, and MpSAHH2 was clustered with SAHHs from methanogen. MpSAHH1 were cloned and expressed heterologously in E. coli strain BL21 (DE3) RIL, and SAH hydrolytic activity was tested with negative result. SAH hydrolysis activity required tetramer SAHH has been discussed. Oligomerization of MpSAHH1 may need for further activity assay. This study demonstrated that halophilic methanogen M. portucalensis FDF1T posses two SAHH from different origins for SAH hydrolysis.
URI: http://hdl.handle.net/11455/23656
其他識別: U0005-0702201201000500
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