Structural and computational studies of the maleate isomerase from Pseudomonas putida S16 reveal a breathing motion wrapping the substrate inside
Structural and computational studies of the maleate isomerase from Pseudomonas putida S16 reveal a breathing motion wrapping the substrate inside
复制标题
对恶臭假单胞菌 S16 马来酸异构酶的结构和计算研究揭示了将底物包裹在内部的呼吸运动
DOI：
10.1111/mmi.12163
复制
发表时间：
2013-03-01
影响因子：
3.6
通讯作者：
Xu, Ping
中科院分区：
文献类型：
--
作者：
Chen, Duoduo;Tang, Hongzhi;Xu, Ping
尼古丁是烟草废料中的一种环境毒物，对人体健康有严重危害。一些细菌，包括假单胞菌属。菌株能够将尼古丁代谢成无毒化合物。最近，恶臭假单胞菌S16中尼古丁降解的吡咯烷途径已经被揭示。马来酸异构酶（Pp-Iso）催化恶臭假单胞菌S16尼古丁降解的最后一步，即顺-反异构化马来酸为富马酸。在这项研究中，我们确定了野生型异构酶本身及其C200 A点突变体与其底物马来酸盐复合物的晶体结构，分辨率分别为2.95埃和2.10埃。我们的结构表明Asn 17和Asn 169在识别顺丁烯二酸酯中起关键作用。令人惊讶的是，我们的结构显示马来酸酯完全包裹在异构酶内。结构的检查促使我们假设2-2环和6-7环具有调节底物/溶剂进入和产物离开的呼吸运动。我们的分子动力学模拟和酶活性测定结果与这一假设完全一致。异构酶可能利用这种呼吸运动来防止溶剂进入活性部位，并阻止非生产性副反应的发生。
Nicotine is an environmental toxicant in tobacco waste, imposing a serious hazard for human health. Some bacteria including Pseudomonas spp. strains are able to metabolize nicotine to non-toxic compounds. The pyrrolidine pathway of nicotine degradation in Pseudomonas putida S16 has recently been revealed. The maleate isomerase (Pp-Iso) catalyses the last step in nicotine degradation of P.putida S16, the cis-trans isomerization of maleate to fumarate. In this study, we determined the crystal structures of both wild type isomerase by itself and its C200A point mutant in complex with its substrate maleate, to resolutions of 2.95 angstrom and 2.10 angstrom respectively. Our structures reveal that Asn17 and Asn169 play critical roles in recognizing the maleate by site-directed mutants' analysis. Surprisingly, our structure shows that the maleate is completely wrapped inside the isomerase. Examination of the structure prompted us to hypothesize that the 2-2 loop and the 6-7 loop have a breathing motion that regulates substrate/solvent entry and product departure. Our results of molecular dynamics simulation and enzymatic activity assay are fully consistent with this hypothesis. The isomerase probably uses this breathing motion to prevent the solvent from entering the active site and prohibit unproductive side reactions from happening.