Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/20583
標題: 受質專一性和鉀離子濃度對嗜鹽性甲烷古生菌相容質甜菜鹼生合成酵素的影響
Substrate and Potassium Effects on Osmolyte Betaine Synthesizing Enzyme - Glycine Sarcosine N, N-dimethylglycine Methyltransferase from Methanohalophilus portucalensis strain FDF1
作者: 王家麒
Wang, Chia-Chi
關鍵字: 古生菌
甜菜鹼
GSDMT
出版社: 植物學系
摘要: 摘要 嗜鹽性甲烷古生菌Methanohalophilus portucalensis是一株可生長在高鹽環境的太古生物,會利用在胞內大量累積甜菜鹼 (glycine betaine)作為相容質 (compatible solutes)來對抗外在的高滲透壓環境。NMR的分析及in vivo和in vitro的相容質甜菜鹼生合成實驗,已證實嗜鹽性甲烷古生菌以S-adenosyl-L- methionine (SAM)為甲基提供者,利用甲基化glycine的方式進行相容質甜菜鹼的生合成。將嗜鹽性甲烷古生菌M. portucalensis strain FDF1的細胞粗萃取液以DEAE-Sephacel陰離子交換樹脂進行純化後,得到一具有能將glycine甲基化成sarcosine活性的glycine N-methyltransferase (GNMT, EC 2.1.1.20)。進一步測試嗜鹽性甲烷古生菌的GNMT的受質專一性時發現,亦具有將sarcosine和dimethylglycine分別甲基化生成dimethylglycine和 glycine betaine的活性,較高等生物及高鹽細菌的GNMT具有較廣的受質使用性。此GNMT會受到鈉和鉀離子濃度不同影響其甲基轉移的能力,生成不同的甲基化產物。在高鉀離子濃度 (800 mM)時傾向生成glycine betaine,低鉀離子濃度 (400 mM)時則傾向生成sarcosine,而鈉離子在高低濃度時則有相反結果。利用2-D電泳及PBE 94 chromatofocusing column可將此嗜鹽性甲烷古生菌的GNMT分為四個分子量相似 (52 kDa),但pI值分別為4.4、5.0、5.2和4.8的胜肽,分別命名為α、β、γ和δ。其中α具有較低的glycine sarcosine dimethylglycine N-methyltransferase (GSDMT)活性,β則具有較高的活性,而γ則僅具有sarcosine dimethylglycine N-methyltransferase (SDMT)的活性。同時α蛋白的GSDMT酵素活性亦受到鉀離子濃度的調控,在低鉀濃度 (≤ 0.4 M)具有較高活性,而γ蛋白的SDMT酵素活性卻只在高鉀濃度時具有較高活性。
Abstract Methanohalophilus portucalensis FDF1 can de novo synthesize and accumulate glycine betaine through threefold methylation of glycine as compatible solutes to overcome the osmotic stress. The activity of glycine N-methyltransferase (GNMT) which formed sarcosine by transferring the methyl group from S-adenosyl-L- methionine (SAM) to glycine was detected by radiometric methods in extracts of M. portucalensis FDF1. The cell extract with GNMT activity was further purified by DEAE-Sephacel ion chromatograph with a 0.1~0.5 M potassium (step or linear) gradient. In addition to the transfer of methyl group from SAM to glycine, experimental results also showed that the GNMT of strain FDF1 was able to transfer the methyl group to sarcosine and N, N-dimethylglycine. GNMT methyltransferring activities on different substrate were effectted by different concentrations of sodium and potassium ions. By 2-D gel and PBE 94 chromatofocusing column, GNMT was further separated into four non-identical subunits (α, β, γ and δ). They have the same molecular weights (52 kDa) and different pI values of 4.4, 5.0, 5.2 and 4.8, respectively. Both the α and β subunits posses the glycine sarcosine N, N-dimethylglycine N-methyltransferase (GSDMT) activity, while the γ subunits only demonstrated the sarcosine N, N-dimethylglycine N-methyltransferase (SNMT) activity. The activities of methyltransferases were regulated by potassium concentrations; the optimal concentration of α, β subunits were around and below 0.4 M, whereas γ subunit was around 0.8 M.
URI: http://hdl.handle.net/11455/20583
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