Merone Luigia, Mandrich Luigi, Rossi Mosè, Manco Giuseppe
Istituto di Biochimica delle Proteine, Consiglio Nazionale delle Ricerche, Via P. Castellino, Napoli, Italy.
Extremophiles. 2005 Aug;9(4):297-305. doi: 10.1007/s00792-005-0445-4. Epub 2005 May 21.
A new gene from the hyperthermophilic archaeon Sulfolobus solfataricus MT4, coding for a putative protein reported to show sequence identity with the phosphotriesterase-related protein family (PHP), was cloned by means of the polymerase chain reaction from the S. solfataricus genomic DNA. In order to analyse the biochemical properties of the protein an overexpression system in Escherichia coli was established. The recombinant protein, expressed in soluble form at 5 mg/l of E. coli culture, was purified to homogeneity and characterized. In contrast with its mesophilic E. coli counterpart that was devoid of any tested activity, the S. solfataricus enzyme was demonstrated to have a low paraoxonase activity. This activity was dependent from metal cations with Co(2+), Mg(2+) and Ni(2+) being the most effective and was thermophilic and thermostable. The enzyme was inactivated with EDTA and o-phenantroline. A reported inhibitor for Pseudomonas putida phosphotriesterase (PTE) had no effect on the S. solfataricus paraoxonase. The importance of a stable paraoxonase for detoxification of chemical warfare agents and agricultural pesticides will be discussed.
从嗜热古菌嗜热栖热菌MT4中克隆出一个新基因，该基因编码一种据报道与磷酸三酯酶相关蛋白家族（PHP）具有序列同一性的假定蛋白，采用聚合酶链反应从嗜热栖热菌基因组DNA中进行克隆。为了分析该蛋白的生化特性，在大肠杆菌中建立了一个过表达系统。以5 mg/l大肠杆菌培养物的可溶形式表达的重组蛋白被纯化至同质并进行了表征。与其缺乏任何测试活性的嗜温大肠杆菌对应物不同，嗜热栖热菌酶被证明具有低对氧磷酶活性。该活性依赖于金属阳离子，其中Co(2+)、Mg(2+)和Ni(2+)最为有效，并且具有嗜热性和热稳定性。该酶被EDTA和邻菲罗啉灭活。一种报道的恶臭假单胞菌磷酸三酯酶（PTE）抑制剂对嗜热栖热菌对氧磷酶没有影响。将讨论稳定对氧磷酶对化学战剂和农用杀虫剂解毒的重要性。
