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    Please use this identifier to cite or link to this item: http://ir.lib.ncu.edu.tw/handle/987654321/71409


    Title: 古生菌嗜酸熱硫化葉菌的乙醯乳酸還原異構酶的晶體結構以及穩定性;Crystal structure and stability of acetohydroxyacid isomeroreductase from hyperthermophilic Sulfolobus acidocaldarius
    Authors: 曾昱尊;Tsen,Yu-Tsuen
    Contributors: 生命科學系
    Keywords: 晶體繞射;嗜酸熱硫化葉菌;乙醯乳酸還原異構酶;Crystal structure;Sulfolobus acidocaldarius;acetohydroxyacid isomeroreductase
    Date: 2016-06-29
    Issue Date: 2016-10-13 13:02:00 (UTC+8)
    Publisher: 國立中央大學
    Abstract: 酵素acetohydroxy acid isomeroreductase (AHIR, EC 1.1.1.86)是在細菌、植物和真菌支鏈型胺基酸合成的第二個酵素,且不會出現在動物細胞中。利用這項動物與其他物種間的差異,可以將酵素AHIR當作抗菌劑或是除草劑的抑制目標,酵素AHIR是再生合成過程中涉及烷基移動以及還原的兩步反應,並且需要輔因子Mg2+以及NAD(P)H的參與。我們同樣在古生菌嗜酸熱硫化葉菌 (Sulfolobus acidocaldarius)中發現這個酵素(Sac_AHIR),此古生菌是在黃石公園的火山口溫泉發現的,屬古泉菌門。我們利用大腸桿菌當宿主細胞,大量表達Sac_AHIR蛋白質,純化並養出蛋白質晶體,以X光繞射收到最高解析度為1.75Å的繞射數據,並用有Se-Met標定的AHIR進行多波長異常散射實驗解出晶體相位,建立蛋白質模型。每個AHIR在自然狀態下形成雙聚體結構,而每個蛋白質在序列上分成兩個部分,N端的Rossmann domain以及C端的knotted domain。兩個knotted domain可以相互作用幫助雙聚體組合,並且形成兩個有功能的活性位。經由比對發現Sac_AHIR的Rossmann domain上的特殊序列β2αB-loop是屬於7個胺基酸類型且偏向親合NADPH作為輔因子。利用圓二色光譜研究可以發現,Sac_AHIR在65°C時會稍微變性直至95°C時會完全變性,而蛋白質的二級結構在pH 3到pH 8之間並沒有顯著的差異。我們推測即使Sac_AHIR在75°C和pH 2的極端溫泉環境,此蛋白質酵素在細胞中依然能保持活性。Sac_AHIR可在高溫與不同酸鹼程度的環境下,保持蛋白質折疊正確,性質相較細菌或植物內的AHIR來得穩定,在生物科技或生質能源上更有應用的潛力;Acetohydroxyacid isomeroreductase (AHIR) is the second key enzyme involved in the branched-chain amino acid biosynthetic pathways which is found in bacteria, fungi and plants but not in animals. This difference in metabolism between animals and microorganisms makes AHIR an attractive target for the development of specific herbicides and antimicrobial agents. Moreover, it is a bifunctional enzyme that catalyzes two reactions, alkyl migration and reduction, and requires Mg2+ and NAD(P)H for activity. Here we present the crystal structure at 1.75 Å resolution of the Sac_AHIR of Sulfolobus acidocaldarius. Sac_AHIR exists as homodimer in solution and each monomer is composed of two types of domains, an N-terminal Rossmann domain and a C-terminal knotted domains. Two intertwined knotted domains are required for formation of the homodimer and two AHIR active sites. Analysis of the amino acid sequences of the Rossmann fold’s β2αB-loop displays that Sac_AHIR has a seven-residue loop, LEREGNS, which prefers using NADPH as the cofactor. Circular dichroism spectrometric analysis showed that Sac_AHIR was denatures slightly at 65°C but unfold completely at 95 °C. The CD spectra of Sac_AHIR was almost identical between at the range of pH 3.0 to 8.0 at 25 °C. The CD results revealed that Sac_AHIR is thermal stable and acid tolerant. Our study suggests that Sac_AHIR might retain activity under the extreme growth environments at temperature 75 °C and pH 2 in solfataric springs.
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