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    Please use this identifier to cite or link to this item: http://ir.lib.ncu.edu.tw/handle/987654321/72578


    Title: 硫化葉菌屬中耐熱酮醇酸還原異構酶之結構性及功能性分析;Structural and Functional Analysis of the Thermotolerant Ketol-Acid Reductoisomerase (KARI) from Sulfolobus species
    Authors: 周志懋;Chou, Chih-Mao
    Contributors: 生命科學系
    Keywords: 酮醇酸還原異構酶;Ketol-Acid Reductoisomerase
    Date: 2016-12-23
    Issue Date: 2017-01-23 16:13:54 (UTC+8)
    Publisher: 國立中央大學
    Abstract: 酮醇酸還原異構酶是參與支鏈型胺基酸生合成的重要酵素,這個酵素只會出現在細菌、古生菌、真菌和植物中,動物則沒有。此酵素催化兩步驟反應,第一步為鎂離子專一性之烷基轉移,在緊接著進行具有 NAD(P)H 和二價金屬 (Mg2+、Mn2+ 及 Co2+) 依賴性之還原反應。大多數被發現的酮醇酸還原異構酶偏好 NADPH,這是因為一般生物是在有氧的環境下生存。在許多文獻中,為了要讓細菌能夠發酵生成支鏈型胺基酸或是將葡萄糖轉化為醇類,利用基因工程的方式將酮醇酸還原異構酶之輔因子專一性改成 NADH。在我們的研究中,從生存在高溫且強酸性之極端環境中的硫化葉屬古生菌中,取得該酵素。在嗜酸熱硫化葉菌中取得 Sac-ilvC;在硫磺礦硫化葉菌中取得 Sso-ilvC-1、Sso-ilvC-2 和 Sso-ilvC-3。在利用 X-ray 蛋白質晶體學以及低溫電子顯微鏡這兩個技術,得到 Sac-ilvC 和 Sso-ilvC-2 之原子級別結構與 Sso-ilvC-3 之奈米級別結構。 再利用光譜學技術對其生化特性進行分析。我們發現 Sac-ilvC 與 Sso-ilvC-1 在序列及特性上都較為接近並且對輔因子具有雙專一性;而 Sso-ilvC-2 與 Sso-ilvC-3 兩者較為相似,會形成十二聚體並且具有 NADH 專一性。酮醇酸異構還原酶聚合化會影響其結構穩定性並提高耐熱能力 ,越是容易聚合之酵素其耐熱能力就越好。至於,硫磺礦硫化葉菌會擁有三個編碼基因的現象,目前推測為該菌若遭遇到更嚴厲之環境,更高溫且含氧量更低,則需要會使用 NADH 且更耐熱之蛋白質。;Ketol-acid reductoisomerase (EC 1.1.1.86) catalyses the second reaction in the biosynthesis of branched-chain amino acids, which is present in plants, fungi, archaea and bacteria, but not in animals. This enzymatic reaction involves an Mg2+-dependent alkyl migration followed by a NADPH- and divalent metal ions-dependent reduction of the 2-keto group. The most of the KARIs characterized to date have been shown to prefer the NADPH cofactor to NADH since these nearly were obtained from the organisms living in aerobic environment. In many case, KARIs switching the cofactor preference to NADH are desirable for industrial applications by genetic engineering, including anaerobic fermentation to produce branched-chain amino acids and some kinds of alcohol. In our study, we obtained four different KARI from Sulfolobus species which are extremophiles living in harsh environments with high temperature and low pH value. Sac-ilvC was gained from Sulfolobus acidocaldarius and Sso-ilvC-1, Sso-ilvC-2 and Sso-ilvC-3 were gained from Sulfolobus solfataricus. Here, we determined the structure of Sso-ilvC-2 by X-ray crystallography (2.5 Å) and cryo-EM (6 Å). The Sso-ilvC-2 forms dodecamer by assembling six dimers as tetrahedral shape. Sso-ilvC-2 and Sso-ilvC-3 are putative NADH-dependent KARI. We supposed S. solfataricus has three KARI encoding genes, which induced the thermoresistan protein that consume NADH when it encountered the much more extreme environment, higher temperature and lower level of oxygen content.
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