低分子量細菌素 Carocin S2 重要胺基酸殘基之活性鑑定以及 3D 結構模型之驗證

朱雅惠

doi:10.6845/NCHU.2013.00499

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低分子量細菌素 Carocin S2 重要胺基酸殘基之活性鑑定以及 3D 結構模型之驗證

Identification of essential amino acid residues of the low-molecular-weight bacteriocin, Carocin S2 and verification of the three-dimensional computational approach

朱雅惠 , Masters　　Advisor：莊敦堯　　

繁體中文 DOI： 10.6845/NCHU.2013.00499

低分子量細菌素；圓二色旋光光譜儀；螢光光譜；模擬結構鑑定； low-molecular-weight bacteriocin, Carocin S2 ； Circular Dichroism spectroscopy ； Intrinsic tryptophan fluorescence

Abstract │ Reference (45) │ Altmetrics

Abstract 〈TOP〉

Pectobacterium carotovorum subsp. carotovorum (Pcc) 為 Pectobacterium 屬，是一種能產生果膠水解酶進而造成許多植物根莖部腐爛的病原菌。低分子量細菌素Carocin S2 是經由 Pcc 菌株 3F-3 所生產，由 CaroS2K 和 CaroS2I 兩個不同分子量的蛋白質所組成。
   CaroS2K 分子量約為 85 kDa ，具抗生活性的殺手蛋白，帶有核醣核酸水解活性； CaroS2I 分子量約為 11 kDa ，與前者形成複合物的免疫蛋白，能抑制細菌素的細胞毒性以避免本身受到傷害。根據實驗室先前研究 Carocin S2數據顯示，已利用 FASTA 蛋白質資料庫對 caroS2K 胺基酸序列進行同源性比對，從中獲得 CaroS2K 的功能區排序資訊: N-terminus 為跨膜功能區；中央功能區則能辨認攻擊目標細胞表面的特定受體，並與之結合，為受體結合功能區；以及 C-terminus 為抗生功能區。除此之外，為了更進一步了解 Carocin S2 及其相關系列突變株的蛋白質折疊結構，在實驗室先前研究中，便使用了 SMART、Phyre 程式等預測 Carocin S2 蛋白的 3D 結構，再以線上軟體 Swiss-PDB Viewer 檢視胺基酸殘基的立體位向。而接下來的研究中，則是將 Carocin S2 單離出帶有 RNase 水解活性的最小區域，爾後進行 in vitro 核醣核酸水解活性試驗。
   本研究中利用圓二色旋光光譜儀 (Circular Dichroism spectroscopy，CD) 和內源性色胺酸螢光光譜儀(Intrinsic tryptophan fluorescence，ITF) 偵測 Carocin S2 及其相關系列突變株，以及 CaroS2-CRD 及其相關系列突變株。我們將其分為四部份探討: Lysine 殘基系列 (CaroS2_K691A、CaroS2_K692A、CaroS2_K695A) ，Histidine 殘基催化中心 (CaroS2_H696A) ，疏水性骨架殘基系列 (CaroS2_Y734A、CaroS2_F760A、CaroS2_W764A) ，以及 tRNAArg biding 殘基 (CaroS2_S762A)。由上述結果，我們發現殘基 H696 可能為重要的蛋白摺疊結構中心而非預測之催化酵素活性中心。此外， 3D 模擬結構和實際結構可能不符，有待後續利用 X-ray 偵測結晶結構。因此我們發現 Carocin S2 可能為新型的細菌素。

Parallel Abstract 〈TOP〉

Pectobacterium carotovorum subsp. carotovorum (Pcc) is a genus of Pectobacterium and a pathogen that produced pectinase causes much stems and roots of plants getting rot. The low-molecular-weight bacteriocin, Carocin S2 is produced from Pcc strain 3F-3 in our laboratory. Carocin S2 is a complex that is composed by two different molecular weight proteins, CaroS2K and CaroS2I.
  The CaroS2K has an antibiotic activity, which we called it as killer protein. It is an 85 kDa protein and has ribonuclease activity. The CaroS2I is an 11 kDa protein, which is an inhibited agent and associated with CaroS2K to form a complex. We also called CaroS2I as immunity protein. Based on previous studies of Carocin S2 in our laboratory, they utilized homolog analysis FASTA protein database with caroS2K gene sequence. The results of CaroS2K’s functional domain indicated that an N-terminal part is transmembrane domain; a central domain is able to identify a specific target cell of surface receptors binding, and C-terminal part is antibiotic domain. In order to understand the folding of Carocin S2 and related mutants, they used SMART、 Phyre program… ect. to predict the 3D structure. Furthermore, they employed the online Swiss-PDB viewer to check the steric structure of residues.
  Here, Circular Dichroism spectroscopy (CD) and Intrinsic tryptophan fluorescence (ITF) were utilized to detect the Carocin S2, the C-terminal RNase domain of Carocin S2 and related mutants in this study. So, they were explored and divided into four parts: Lysine residues series (CaroS2_K691A、CaroS2_K692A、CaroS2_K695A), catalytic center of Histidine residues (CaroS2_H696A), hydrophobic skeleton of residues series (CaroS2_Y734A、CaroS2_F760A、CaroS2_W764A), and tRNAArg biding residues (CaroS2_S762A). From the results, we found that residues H696 may be an important structure center for protein folding, but not the predicted catalytic center. Besides, the result of 3D computational approach is not consistent with the real structure. The crystal structure will be detected by X-ray in the future. Therefore, it seems that Carocin S2 could be a new type of bacteriocin.

Reference ( 45 ) 〈TOP〉

1. Gardan, L., Gouy, C., Christen, R., & Samson, R. (2003). Elevation of three subspecies of Pectobacterium carotovorum to species level: Pectobacterium atrosepticum sp. nov., Pectobacterium betavasculorum sp. nov. and Pectobacterium wasabiae sp. nov. International journal of systematic and evolutionary microbiology, 53(2), 381-391.
連結：
2. Nguyen, H. A., Tomita, T., Hirota, M., Kaneko, J., Hayashi, T., & Kamio, Y. (2001). DNA inversion in the tail fiber gene alters the host range specificity of carotovoricin Er, a phage-tail-like bacteriocin of phytopathogenic Erwinia carotovora subsp. carotovora Er. Journal of bacteriology, 183(21), 6274-6281.
連結：
3. LUND, B. M., & Wyatt, G. M. (1973). The nature of reducing compounds formed from sucrose by Erwinia carotovora var. atroseptica. Journal of General Microbiology, 78(2), 331-336.
連結：
5. Malcolmson, J. F. (1959). A study of Erwinia isolates obtained from soft rots and blackleg of potatoes. Transactions of the British Mycological Society, 42(2), 261-269.
連結：
6. Phillips, J. A., & Kelman, A. (1982). Direct fluorescent antibody stain procedure applied to insect transmission ofErwinia carotovora. Phytopathology, 72, 898-901.
連結：

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