青海弧菌荧光素酶蛋白三维结构的分子模拟研究

Molecular Modeling Study on the Three-dimensional Structure of the Luciferase Protein in Vibrio-qinghaiensis sp.-Q67

基于荧光素酶催化发光的生物发光技术在化学、生命与环境科学等领域得到广泛应用.部分萤火虫和发光细菌的荧光素酶晶体三维结构已经解析出来.青海弧菌(Vibrio qinghaiensis sp.-Q67,简称Q67)是从青海湖青海裸鲤中提取的一种新型淡水发光细菌,已用于化学品的生物检测及毒性评价.然而,其催化发光过程中最重要的荧光素酶三维结构尚未解析,阻碍了化学品对Q67产生毒性的机理研究进程.本工作通过异源二聚体同源建模与分子动力学模拟方法构建了Q67荧光素酶的三维结构.研究结果表明,Q67荧光素酶蛋白有α和β两个多肽亚基,范德华力是维持α/β稳定结构的主要作用力,而α/β亚基携带的净电荷产生的静电相互作用不利于体系稳定.缔合的专一性则由α/β亚基接触面上的极性基团之间的氢键实现.底物黄素单核苷酸与Q67荧光素酶的结合位点在α亚基活性口袋中,β亚基有利于保持α亚基活性口袋的稳定性.

Bioluminescence technique derived from the luciferase-based catalization reactions has been widely used in chemistry, biological assay and environmental science. The three-dimensional crystal structures of luciferase proteins in some firefly and luminescent bacteria were elucidated. Vibrio qinghaiensis sp.-Q67 （Q67）, one of freshwater luminescent bacteria which was extracted from Gymnocypris przewalskii in Qinghai lake, has been used in biological assay and toxicity evaluation of many chemicals. However, the crystal structure of the luciferase （the most important catalyzer to bioluminescent） in Q67 is still not established, which hinders the process of the study on the molecular mechanism of toxicities of chemicals to Q67. In this study, the three dimensional structure of bacterial luciferase in Q67 was constructed by using the heterodimeric homol- ogy modeling combined with the molecular dynamics simulation which were performed with explicit TIP3P water. The simulation system was equilibrated at 4 ns, and was prolonged for another 4 ns for extracting the equilibrium trajectories at the 8th ns. The stability of the system was monitored through the convergences of energy, temperature, and global root mean square deviation （RMSD）. The ptraj modules in the AMBER software were used to analyze hydrogen bond occupancy between ct and 13 subunit. And then, the molecular mechanics generalized born surface area method was applied to identity/ critical amino acids of the u and 13 subunits that interact with each other during the native heterotetrameric structure formation. It was shown that the luciferase in Q67 is a heterdimer including two polypeptide subunits （α and β） and the stabilization of this heterodimer was mainly determined by the van der waals force. The specificity of association is realized by hydrogen bonds formed between subunits. However, the electrostatic interaction from the net charge on ct and 13 subunit is unfavorable to the stability of the dimer. The active sites of flavin mononucleotide binding to the luciferase in Q67 are located in the active pocket ofα subunit. The β subunit is helpful to keep the structural stability of the active sites on the α subunit.