Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22235
標題: 噬菌體 P1201之 ant4 基因在Corynebacterium glutamicum NCHU 87078 之生理角色及Proteus mirabilis BCRC 10725 之離胺酸消旋酵素之生化特性
Physiological role of corynephage P1201 ant4 gene in Corynebacterium glutamicum NCHU 87078 and biochemical characterization of a novel lysine racemase from Proteus mirabilis BCRC 10725
作者: 管宜家
Kuan, Yi-Chia
關鍵字: Corynebacterium glutamicum
基因體
ant4
載體
出版社: 分子生物學研究所
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摘要: (1)Lytic phage P1201 was isolated from Corynebacterium glutamicum NCHU 87078 that was infected by phage during an industrial fermentation for glutamic acid production. P1201 genome contains an ant4 gene, its biological function remains unclear. The Ant4 protein contains two conserved domains: N terminal bro-N domain and C-terminal kilA-C domain. The full-length and truncated ant4 genes, designated ant4, bro-N domain and kilA-C domain were cloned and expressed in Escherichia coli NovaBlue and C. glutamicum. Two Ant4 proteins with different N-terminus were expressed in E. coli and C. glutamicum harboring ant4 gene. Growth inhibition and cell death were observed for the C. glutamicum and E. coli cells expressing ant4 gene. Realtime-PCR and Western blot analysis indicated that the expression of ant4 gene occurs in the early stage of phage infection. Microarray analysis the expression levels of revealed that 218 genes were significantly changed, when the ant4 gene was expressed in C. glutamicum. The transcription of ion transportation related genes was increased in the cells expressing ant4, while the expression level of genes related to energy generation and protein translation were decreased. The data from realtime-PCR assay indicated that Ant4 inhibited the expression of the genes related to carbohydrate and fatty acid metabolism, including aceA, aceB, ilvB, fas-IA and fas-IB. Pull-down experiments and immunoprecipitation assay revealed Ant4 interacted with transcriptional regulators including SugR, RamA, RamB, and LexA of C. glutamicum NCHU 87078, and the C-terminal kilA-C domain is required for protein-protein interaction. EMSA indicated that Ant4 could enhance the binding of RamA and LexA to DNA fragment containing their specific binding sites. Ant4 could facililate formation of loop comformation for ace-P. Uniformly and non-specifically binding of Ant4 to the aceA-P DNA fragment was demonstrated using atomic force microscopy. However, specific binding of Ant4 to the RamA binding site coulded be found in the presence of the tramscriptional regulator RamA. Our data suggested the Ant4 protein might be a global transcriptional regulator after corynephage P1201 infection to suppress the metabolism in host. (2)A lysine racemase gene (lyr) that consisted of an open reading frame of 1224-bp and encoded a protein with a calculated molecular mass of 45 kDa was cloned from the Proteus mirabilis BCRC10725 and expressed in Escherichia coli BL21(DE3). The purified His6-tagged Lyr was most active towards lysine, exhibiting a specific activity of 2828±97 U/mg. This enzyme also racemized arginine with a specific activity of 568±28 U/mg but not other amino acids. The optimal conditions for Lyr activity to L-lysine were pH 8.0-9.0 and 50˚C. The racemization activity of Lyr was completely inhibited by 5 mM hydroxylamine and was partially restored by the addition of pyridoxal 5'-phosphate. The S394 residue of Lyr was subjected to site-directed mutagenesis. The arginine racemization activities of the S394Y, S394N, S394C and S394T variant proteins were increased by 1.5-1.8 fold compared to the wild-type Lyr, indicating that the S394 residue played a crucial role in determining the preference of Lyr to lysine and arginine.
(1)利用 Corynebacterium glutamicum NCHU 87078 進行味精醱酵時,有時會發生敗槽,前人研究中由敗槽醱酵液內分離出溶裂性噬菌體 corynephage P1201,其基因體中的 ant4 基因之生理角色,仍無文獻報導。Ant4 具有兩個 domain,N 端 bro-N domain 屬於 bro family,可能為結合 DNA 的 domain,而 C 端則比對到 prophage 的 kilA-C domain。從 C. glutamicum NCHU 87078 中選殖出 ant4 基因、bro-N domain 及 kilA-C domain DNA 片段,分別將三個 DNA 片段構築於表現載體上,進行大量表現並純化蛋白,發現 ant4 於 C. glutamicum 或 E. col 菌體內表現時,會轉譯出兩種 N 端有差異的蛋白質,並造成 C. glutamicum 和 E. coli 停止生長。以 realtime-PCR 及 Western blot 偵測 corynephage P1201 感染 C. glutamicum NCHU 87078 時 ant4 的表現情形,發現 ant4 屬於病毒感染的早期基因。利用核酸微矩陣 (microarray) 分析表現 ant4 基因的 C. glutamicum NCHU 87078,發現有 218 個基因和對照組有顯著差異。其中與離子輸送相關之基因在ant4基因表現時,表現量上升,而大部分與能量產生及轉譯有關之基因則表現量下降。進一步以 realtime-PCR 分析確認,發現 RamA 所調控之酒精代謝及醋酸代謝之相關基因 (aceA及aceB) 表現量下降,與支鏈型胺基酸 (ilvB) 及脂肪酸合成相關的基因 (fas-IA及fas-IB) 之表現量也下降,推測前述基因表現之下降,可能與 Ant4 抑制 C. glutamicum NCHU 87078 的生長有關。利用 Pull-down assay 及免疫沉澱法分析蛋白質之交互作用,發現 Ant4 會與 C. glutamicum NCHU 87078 的轉錄調控因子: SugR 、 RamA 、 RamB 及 LexA 產生交互作用,並確認 Ant4 蛋白 C 端的 kilA-C domain 為參與蛋白質交互作用的主要區域,而 N 端的bro-N domain 為與 DNA 結合的主要區域。利用 EMSA 分析結果也顯示, Ant4 會促進 RamA 及 LexA 與其專一性 DNA 結合的能力。以原子力顯微鏡觀察,發現 Ant4 單獨存在時會非專一性的結合在整條 aceA-P DNA 片段上。但是在 RamA 轉錄調控因子存在下,Ant4 會藉由與 RamA 的相互作用形成一個 Ant4/RamA/aceA-P 的聚合結構,結合在 RamA 的啟動子上,使 aceA 基因無法進行轉錄。綜上所述,Ant4 可能扮演著 corynephage P1201 感染宿主後調控代謝基因表現的廣泛性轉錄調節者 (global transcriptional regulator)。 (2)從Proteus mirabilis BCRC 10725中選殖一完整的ORF並將其命名為lyr,此基因全長為1224 bp,可以轉譯出預估分子量為45 kDa的蛋白質。將lyr基因於E. coli BL21(DE3)中表現,純化帶有His6-tag的Lyr酵素,可催化lysine 的消旋反應,比活性為2828 ± 97 U/mg;此酵素對arginine 也具有催化的活性,其比活性為568 ±28 U/mg,但對於其他胺基酸則不具有消旋作用。Lyr的最適反應pH及溫度分別為8.0-9.0 及50 ℃,其活性不受離子的影響。將Lyr以hydroxylamine處理後會失去lysine racemase活性,再添加pyridoxal 5`-phosphate (PLP) 後可恢復部份活性,證實其反應需要PLP。將Lyr第394個胺基酸的serine做定點突變,發現S394Y、 S394N、 S394C及S394T 對arginine的轉換率可提升約1.5-1.8倍,由結果可知S394在Lyr對lysine 及arginine之基質選擇性上扮演著重要角色。
URI: http://hdl.handle.net/11455/22235
其他識別: U0005-2109201112584900
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