Guo Xiaoliang, Li Zengru, Jiang Qinqin, Cheng Cai, Feng Yu, He Yanlin, Zuo Lingzi, Rao Li, Ding Wei, Feng Lingling
Key Laboratory of Pesticide and Chemical Biology (CCNU), Ministry of Education, College of Chemistry, Central China Normal University, Wuhan, China.
The Institute of Physics, Chinese Academy of Sciences, Beijing, China.
Front Microbiol. 2023 Feb 17;14:1057264. doi: 10.3389/fmicb.2023.1057264. eCollection 2023.
Removing microcystins (MCs) safely and effectively has become an urgent global problem because of their extremely hazardous to the environment and public health. Microcystinases derived from indigenous microorganisms have received widespread attention due to their specific MC biodegradation function. However, linearized MCs are also very toxic and need to be removed from the water environment. How MlrC binds to linearized MCs and how it catalyzes the degradation process based on the actual three-dimensional structure have not been determined. In this study, the binding mode of MlrC with linearized MCs was explored using a combination of molecular docking and site-directed mutagenesis methods. A series of key substrate binding residues, including E70, W59, F67, F96, S392 and so on, were identified. Sodium dodecane sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze samples of these variants. The activity of MlrC variants were measured using high performance liquid chromatography (HPLC). We used fluorescence spectroscopy experiments to research the relationship between MlrC enzyme (E), zinc ion (M), and substrate (S). The results showed that MlrC enzyme, zinc ion and substrate formed E-M-S intermediates during the catalytic process. The substrate-binding cavity was made up of N and C-terminal domains and the substrate-binding site mainly included N41, E70, D341, S392, Q468, S485, R492, W59, F67, and F96. The E70 residue involved in both substrate catalysis and substrate binding. In conclusion, a possible catalytic mechanism of the MlrC enzyme was further proposed based on the experimental results and a literature survey. These findings provided new insights into the molecular mechanisms of the MlrC enzyme to degrade linearized MCs, and laid a theoretical foundation for further biodegradation studies of MCs.
由于微囊藻毒素（MCs）对环境和公众健康具有极大危害，安全有效地去除微囊藻毒素已成为一个紧迫的全球性问题。源自本土微生物的微囊藻毒素酶因其特定的MC生物降解功能而受到广泛关注。然而，线性化的MCs毒性也很强，需要从水环境中去除。基于实际三维结构，MlrC如何与线性化的MCs结合以及如何催化降解过程尚未确定。在本研究中，结合分子对接和定点诱变方法探索了MlrC与线性化MCs的结合模式。鉴定出了一系列关键的底物结合残基，包括E70、W59、F67、F96、S392等。使用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析这些变体的样品。使用高效液相色谱（HPLC）测量MlrC变体的活性。我们通过荧光光谱实验研究了MlrC酶（E）、锌离子（M）和底物（S）之间的关系。结果表明，MlrC酶、锌离子和底物在催化过程中形成了E-M-S中间体。底物结合腔由N端和C端结构域组成，底物结合位点主要包括N41、E70、D341、S392、Q468、S485、R492、W59、F67和F96。E70残基既参与底物催化又参与底物结合。总之，基于实验结果和文献调研进一步提出了MlrC酶可能的催化机制。这些发现为MlrC酶降解线性化MCs的分子机制提供了新的见解，并为MCs的进一步生物降解研究奠定了理论基础。
