Kold David, Dauter Zbigniew, Laustsen Anne K, Brzozowski Andrzej M, Turkenburg Johan P, Nielsen Anders D, Koldsø Heidi, Petersen Evamaria, Schiøtt Birgit, De Maria Leonardo, Wilson Keith S, Svendsen Allan, Wimmer Reinhard
Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, Sohngaardsholmsvej 49, DK-9000, Aalborg, Denmark.
Protein Sci. 2014 Aug;23(8):1023-35. doi: 10.1002/pro.2489. Epub 2014 Jun 16.
The interaction of lipolytic enzymes with anionic surfactants is of great interest with respect to industrially produced detergents. Here, we report the interaction of cutinase from the thermophilic fungus Humicola insolens with the anionic surfactant SDS, and show the enzyme specifically binds a single SDS molecule under nondenaturing concentrations. Protein interaction with SDS was investigated by NMR, ITC and molecular dynamics simulations. The NMR resonances of the protein were assigned, with large stretches of the protein molecule not showing any detectable resonances. SDS is shown to specifically interact with the loops surrounding the catalytic triad with medium affinity (Ka ≈ 10(5) M(-1) ). The mode of binding is closely similar to that seen previously for binding of amphiphilic molecules and substrate analogues to cutinases, and hence SDS acts as a substrate mimic. In addition, the structure of the enzyme has been solved by X-ray crystallography in its apo form and after cocrystallization with diethyl p-nitrophenyl phosphate (DNPP) leading to a complex with monoethylphosphate (MEP) esterified to the catalytically active serine. The enzyme has the same fold as reported for other cutinases but, unexpectedly, esterification of the active site serine is accompanied by the ethylation of the active site histidine which flips out from its usual position in the triad.
就工业生产的洗涤剂而言，脂解酶与阴离子表面活性剂之间的相互作用备受关注。在此，我们报道了嗜热真菌腐质霉角质酶与阴离子表面活性剂十二烷基硫酸钠（SDS）之间的相互作用，并表明在非变性浓度下该酶特异性结合单个SDS分子。通过核磁共振（NMR）、等温滴定量热法（ITC）和分子动力学模拟研究了蛋白质与SDS的相互作用。对该蛋白质的NMR共振峰进行了归属，发现蛋白质分子的大片段未显示任何可检测到的共振峰。结果表明，SDS以中等亲和力（Ka≈10⁵ M⁻¹）与围绕催化三联体的环特异性相互作用。其结合模式与之前观察到的两亲性分子和底物类似物与角质酶的结合模式非常相似，因此SDS起到了底物模拟物的作用。此外，通过X射线晶体学解析了该酶的无配体形式结构以及与对硝基苯磷酸二乙酯（DNPP）共结晶后形成的结构，后者形成了一个活性丝氨酸被单乙酯磷酸（MEP）酯化的复合物。该酶具有与其他角质酶报道的相同折叠结构，但出乎意料的是，活性位点丝氨酸的酯化伴随着活性位点组氨酸的乙基化，组氨酸从其在三联体中的通常位置翻转出来。
