Manco G, Adinolfi E, Pisani F M, Ottolina G, Carrea G, Rossi M
Istituto di Biochimica delle Proteine ed Enzimologia, CNR, Via Marconi 10, 80125 Naples, Italy.
Biochem J. 1998 May 15;332 ( Pt 1)(Pt 1):203-12. doi: 10.1042/bj3320203.
We previously purified a new esterase from the thermoacidophilic eubacterium Bacillus acidocaldarius whose N-terminal sequence corresponds to an open reading frame (ORF3) reported to show homology with the mammalian hormone-sensitive lipase (HSL)-like group of the esterase/lipase family. To compare the biochemical properties of this thermophilic enzyme with those of the homologous mesophilic and psychrophilic members of the HSL group, an overexpression system in Escherichia coli was established. The protein, expressed in soluble and active form at 10 mg/l E. coli culture, was purified to homogeneity and characterized biochemically. The enzyme, a 34 kDa 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-exanoate with Km and kcat values of 11+/-2 microM (mean+/-S.D., n=3) and 6610+/-880 s-1 (mean+/-S.D., n=3) respectively at 70 degreesC and pH7.1. In spite of relatively high sequence identity with the mammalian HSLs, the psychrophilic Moraxella TA144 lipase 2 and the human liver arylacetamide deacetylase, no lipase or amidase activity was detected. A series of substrates were tested for enantioselectivity. Substantial enantioselectivity was observed only in the resolution of (+/-)-3-bromo-5-(hydroxymethyl)-Delta2-isoxazoline, where the (R)-product was obtained with an 84% enantiomeric excess at 36% conversion. The enzyme was also able to synthesize acetyl esters when tested in vinyl acetate and toluene. Inactivation by diethylpyrocarbonate, diethyl-p-nitrophenyl phosphate, di-isopropylphosphofluoridate (DFP) and physostigmine, as well as labelling with [3H]DFP, supported our previous suggestion of a catalytic triad made up of Ser-His-Asp. The activity-stability-temperature relationship is discussed in relation to those of the homologous members of the HSL group.
我们之前从嗜热嗜酸真细菌嗜酸热芽孢杆菌中纯化出一种新的酯酶，其N端序列与一个开放阅读框（ORF3）相对应，据报道该开放阅读框与酯酶/脂肪酶家族中哺乳动物激素敏感性脂肪酶（HSL）样基团具有同源性。为了比较这种嗜热酶与HSL基团中同源嗜温和嗜冷成员的生化特性，我们在大肠杆菌中建立了一个过表达系统。该蛋白以可溶且有活性的形式在10 mg/L的大肠杆菌培养物中表达，经纯化后达到同质，并进行了生化特性分析。基于底物特异性和抑制剂的作用，该酶是一种34 kDa的单体蛋白，被证明是一种B'型羧酸酯酶（EC 3.1.1.1）。在所测试的对硝基苯基（PNP）酯中，最佳底物是PNP-己酸酯，在70℃和pH7.1条件下，其Km和kcat值分别为11±2 μM（平均值±标准差，n = 3）和6610±880 s-1（平均值±标准差，n = 3）。尽管与哺乳动物HSL、嗜冷的莫拉氏菌TA144脂肪酶2和人肝脏芳基乙酰胺脱乙酰酶具有较高的序列同一性，但未检测到脂肪酶或酰胺酶活性。我们测试了一系列底物的对映选择性。仅在拆分（±）-3-溴-5-（羟甲基）-Δ2-异恶唑啉时观察到显著的对映选择性，在此过程中，在36%的转化率下获得了对映体过量84%的（R）-产物。在乙酸乙烯酯和甲苯中进行测试时，该酶也能够合成乙酸酯。焦碳酸二乙酯、对硝基苯基磷酸二乙酯、二异丙基氟磷酸酯（DFP）和毒扁豆碱的失活作用，以及用[3H]DFP进行标记，支持了我们之前提出的由Ser-His-Asp组成催化三联体的观点。我们结合HSL基团同源成员的活性-稳定性-温度关系对其进行了讨论。
