Kyeong Hyun-Ho, Kim Jin-Hyun, Kim Hak-Sung
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, 305-701, Korea.
Appl Microbiol Biotechnol. 2015 Jun;99(11):4735-42. doi: 10.1007/s00253-014-6304-4. Epub 2014 Dec 31.
N-Acyl homoserine lactone (AHL) is a major quorum-sensing signaling molecule in many bacterial species. Quorum-quenching (QQ) enzymes, which degrade such signaling molecules, have attracted much attention as an approach to controlling and preventing bacterial virulence and pathogenesis. However, naturally occurring QQ enzymes show a broad substrate spectrum, raising the concern of unintentionally attenuating beneficial effects by symbiotic bacteria. Here we report the rational design of acyl homoserine lactonase with high substrate specificity. Through docking analysis, we identified three key residues which play a key role in the substrate preference of the enzyme. The key residues were changed in a way that increases hydrophobic contact with a substrate having a short acyl chain (C4-AHL) while generating steric clashes with that containing a long acyl chain (C12-AHL). The resulting mutants exhibited a significantly shifted preference toward a substrate with a short acyl chain. Molecular dynamics simulations suggested that the mutations affect the behavior of a flexible loop, allowing tighter binding of a substrate with a short acyl chain.
N-酰基高丝氨酸内酯（AHL）是许多细菌物种中主要的群体感应信号分子。降解此类信号分子的群体淬灭（QQ）酶作为一种控制和预防细菌毒力及发病机制的方法已备受关注。然而，天然存在的QQ酶显示出广泛的底物谱，这引发了人们对共生细菌无意减弱有益作用的担忧。在此，我们报告了具有高底物特异性的酰基高丝氨酸内酯酶的合理设计。通过对接分析，我们确定了三个在酶的底物偏好中起关键作用的关键残基。这些关键残基的改变方式是增加与具有短酰基链（C4-AHL）的底物的疏水接触，同时与含有长酰基链（C12-AHL）的底物产生空间冲突。所得突变体对具有短酰基链的底物表现出明显改变的偏好。分子动力学模拟表明，这些突变影响了一个柔性环的行为，使得具有短酰基链的底物能够更紧密地结合。
