Wei Jia, Huang Feiyu, Feng Hai, Massey Isaac Yaw, Clara Tezi, Long Dingxin, Cao Yi, Luo Jiayou, Yang Fei
Hunan Provincial Key Laboratory of Clinical Epidemiology, Xiangya School of Public Health, Central South University, Changsha, China.
Hunan Province Key Laboratory of Typical Environmental Pollution and Health Hazards, School of Public Health, University of South China, Hengyang, China.
Front Microbiol. 2021 Mar 30;12:646084. doi: 10.3389/fmicb.2021.646084. eCollection 2021.
Microcystins (MCs) are extremely hazardous to the ecological environment and public health. How to control and remove MCs is an unsolved problem all over the world. Some microbes and their enzymes are thought to be effective in degrading MCs. Microcystinase can linearize microcystin-leucine-arginine (MC-LR) a specific locus. However, linearized MC-LR is also very toxic and needs to be removed. How linearized MC-LR was metabolized by linearized-microcystinase, especially how linearized-microcystinase binds to linearized MC-LR, has not been defined. A combination of experiments and computer simulation was applied to explore the characterization and molecular mechanisms for linearized MC-LR degraded by linearized-microcystinase. The purified linearized-microcystinase was obtained by recombinant overexpressing. The concentration of linearized MC-LR was detected by high-performance liquid chromatography, and linearized MC-LR degradation products were analyzed by the mass spectrometer. Homology modeling was used to predict the structure of the linearized-microcystinase. Molecular docking techniques on the computer were used to simulate the binding sites of linearized-microcystinase and linearized MC-LR. The purified linearized-microcystinase was obtained successfully. The linearized-microcystinase degraded linearized MC-LR to tetrapeptide efficiently. The second structure of linearized-microcystinase consisted of many alpha-helices, beta-strands, and colis. Linearized-microcystinase interacted the linearized MC-LR with hydrogen bond, hydrophobic interaction, electrostatic forces, and the Van der Waals force. This study firstly reveals the characterization and specific enzymatic mechanism of linearized-microcystinase for catalyzing linearized MC-LR. These findings encourage the application of MC-degrading engineering bacteria and build a great technique for MC-LR biodegradation in environmental engineering.
微囊藻毒素（MCs）对生态环境和公众健康危害极大。如何控制和去除微囊藻毒素是全球尚未解决的问题。一些微生物及其酶被认为对降解微囊藻毒素有效。微囊藻毒素酶可使微囊藻毒素 - 亮氨酸 - 精氨酸（MC - LR）在特定位点线性化。然而，线性化的MC - LR毒性也很强，需要被去除。线性化的MC - LR如何被线性化微囊藻毒素酶代谢，尤其是线性化微囊藻毒素酶如何与线性化的MC - LR结合，尚未明确。本研究采用实验与计算机模拟相结合的方法，探索线性化微囊藻毒素酶降解线性化MC - LR的特性及分子机制。通过重组过表达获得纯化的线性化微囊藻毒素酶。采用高效液相色谱法检测线性化MC - LR的浓度，用质谱仪分析线性化MC - LR的降解产物。利用同源建模预测线性化微囊藻毒素酶的结构。运用计算机分子对接技术模拟线性化微囊藻毒素酶与线性化MC - LR的结合位点。成功获得了纯化的线性化微囊藻毒素酶。线性化微囊藻毒素酶能有效地将线性化MC - LR降解为四肽。线性化微囊藻毒素酶的二级结构由许多α - 螺旋、β - 折叠和无规卷曲组成。线性化微囊藻毒素酶通过氢键、疏水相互作用、静电力和范德华力与线性化MC - LR相互作用。本研究首次揭示了线性化微囊藻毒素酶催化线性化MC - LR的特性及具体酶促机制。这些发现促进了微囊藻毒素降解工程菌的应用，并为环境工程中MC - LR的生物降解构建了一项重要技术。
