Cloning, overexpression, and properties of a new thermophilic and thermostable esterase with sequence similarity to hormone-sensitive lipase subfamily from the archaeon Archaeoglobus fulgidus
Cloning, overexpression, and properties of a new thermophilic and thermostable esterase with sequence similarity to hormone-sensitive lipase subfamily from the archaeon Archaeoglobus fulgidus
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DOI：
10.1006/abbi.1999.1497
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发表时间：
2000-01-01
影响因子：
3.9
通讯作者：
Rossi, M
中科院分区：
文献类型：
--
作者：
Manco, G;Giosuè, E;Rossi, M
据报道，来自超嗜热古菌Archaeoglobus fulgidus的一个新酯酶基因与酯酶/脂肪酶家族的哺乳动物激素敏感脂肪酶（HSL）样组具有同源性，该基因是通过聚合酶链式反应从A. fulgidus基因组中克隆出来的。为了将这种推定的超嗜热酶的生化特性与 HSL 组的同源嗜热成员，即脂环酸芽孢杆菌（以前称为芽孢杆菌）酸热酯酶 2 (EST2) 的生化特性进行比较，在大肠杆菌中建立了过表达系统。重组蛋白以可溶性活性形式在大肠杆菌培养物中以 20 毫克/升的浓度表达，经纯化至均质并进行表征。该酶是一种 35.5-MDa 单体蛋白，根据底物特异性和抑制剂的作用，被证明是 B" 型羧酸酯酶 (EC 3.1.1.1)。在测试的对硝基苯 (PNP) 酯中，最佳底物是 PNP.己酸酯，K-m 和 k(cat) 值为 11 +/- 3 mu M（平均值 +/- SD，n = 3）和 1014 +/- 38 s(-1)（平均值 +/- SD，n = 3），分别在 70 摄氏度和 pH 7.1 下，通过焦碳酸二乙酯、苯甲基磺酰氟、氟膦酸二异丙酯 (DFP) 和毒扁豆碱进行灭活，以及用 [H-3]DFP 进行标记，支持了我们之前关于由以下物质组成的催化三联体的建议。 Ser(160)-His(285)-Asp(255) 与热稳定性 A.acidocaldarius EST2 的序列一致性为 42.5%。通过频域荧光测定和各向异性衰减研究了两种蛋白质的构象动力学，并讨论了活性/稳定性/温度关系。
A new esterase gene from the hyperthermophilic archaeon Archaeoglobus fulgidus, reported to show homology with the mammalian hormone-sensitive lipase (HSL)-like group of the esterase/lipase family, was cloned by means of the polymerase chain reaction from the A. fulgidus genome. In order to compare the biochemical properties of this putative hyperthermophilic enzyme with those of the homologous, thermophilic member of HSL group, namely Alicyclobacillus (formerly Bacillus) acidocaldarius esterase 2 (EST2), an overexpression system in Escherichia coli was established. The recombinant protein, expressed in soluble and active form at 20 mg/liter of E, coli culture, was purified to homogeneity and characterized. The enzyme, a 35.5-MDa monomeric protein, was demonstrated to be a B "-type carboxylesterase (EC 3.1.1.1) on the basis of substrate specificity and the action of inhibitors. Among the p-nitrophenyl (PNP) esters tested the best substrate was PNP.hexanoate with K-m and k(cat) values of 11 +/- 3 mu M (mean +/- SD, n = 3) and 1014 +/- 38 s(-1) (mean +/- SD, n = 3), respectively, at 70 degrees C and pH 7.1. Inactivation by diethylpyrocarbonate, phenylmethyl-sulfonylfluoride, diisopropylfosfofluoridate (DFP), and physostigmine, as well as labeling with [H-3]DFP, supported our previous suggestion of a catalytic triad made up of Ser(160)-His(285)-Asp(255). The sequence identity with the thermostable A. acidocaldarius EST2 was 42.5%. The enzyme proved to be much more stable than its Alicyclobacillus counterpart. The conformational dynamics of the two proteins were investigated by frequency-domain fluorometry and anisotropy decay and the activity/stability/temperature relationship was discussed. (C) 2000 Academic Press.