Interdomain Hydrophobic Interactions Modulate the Thermostability of Microbial Esterases from the Hormone-Sensitive Lipase Family
Interdomain Hydrophobic Interactions Modulate the Thermostability of Microbial Esterases from the Hormone-Sensitive Lipase Family
复制标题
域间疏水相互作用调节激素敏感脂肪酶家族微生物酯酶的热稳定性
DOI：
10.1074/jbc.m115.646182
复制
发表时间：
2015-04-24
影响因子：
4.8
通讯作者：
Zhang, Xi-Ying
中科院分区：
文献类型：
--
作者：
Li, Ping-Yi;Chen, Xiu-Lan;Zhang, Xi-Ying
微生物脂肪酶（HSL）含有CAP结构域和催化结构域。然而，CAP结构域如何与催化结构域相互作用以维持微生物HSL的稳定性仍不清楚。在这里，我们从海洋沉积物宏基因组文库中分离出HSL酯酶E40。E40在45 ℃时活性最高，且不耐热，在40 ℃时半衰期仅为2 min，这可能是E40对长期低温沉积环境的一种适应。对E40的结构进行解析，研究其热不稳定性。结构分析表明，E40缺乏环1的CAP结构域和α 7的催化结构域之间的域间疏水相互作用相比，其热稳定的同源物。突变分析表明，在α 7中引入疏水残基Trp（202）和Phe（203）显著提高了E40的稳定性，并且在环1中进一步引入疏水残基使得E40更耐热，因为形成了结构域间的疏水相互作用。总之，结果表明，环1和α 7之间的结构域间疏水相互作用的缺乏导致E40的热不稳定性。此外，对E40和其他热不稳定和热稳定的HSL的结构的比较分析表明，环1和α 7之间的结构域间疏水相互作用是微生物HSL热稳定性的关键因素。因此，本研究不仅阐明了导致E40热不稳定性的结构因素，而且揭示了HSL热稳定性的结构决定因素。
Microbial hormone-sensitive lipases (HSLs) contain a CAP domain and a catalytic domain. However, it remains unclear how the CAP domain interacts with the catalytic domain to maintain the stability of microbial HSLs. Here, we isolated an HSL esterase, E40, from a marine sedimental metagenomic library. E40 exhibited the maximal activity at 45 degrees C and was quite thermolabile, with a half-life of only 2 min at 40 degrees C, which may be an adaptation of E40 to the permanently cold sediment environment. The structure of E40 was solved to study its thermolability. Structural analysis showed that E40 lacks the interdomain hydrophobic interactions between loop 1 of the CAP domain and alpha 7 of the catalytic domain compared with its thermostable homologs. Mutational analysis showed that the introduction of hydrophobic residues Trp(202) and Phe(203) in alpha 7 significantly improved E40 stability and that a further introduction of hydrophobic residues in loop 1 made E40 more thermostable because of the formation of interdomain hydrophobic interactions. Altogether, the results indicate that the absence of interdomain hydrophobic interactions between loop 1 and alpha 7 leads to the thermolability of E40. In addition, a comparative analysis of the structures of E40 and other thermolabile and thermostable HSLs suggests that the interdomain hydrophobic interactions between loop 1 and alpha 7 are a key element for the thermostability of microbial HSLs. Therefore, this study not only illustrates the structural element leading to the thermolability of E40 but also reveals a structural determinant for HSL thermostability.