Yoneda Kazunari, Yoshioka Misa, Sakuraba Haruhiko, Araki Tomohiro, Ohshima Toshihisa
Department of Bioscience, School of Agriculture, Tokai University, 9-1-1 Toroku, Higashi-ku, Kumamoto-shi, Kumamoto 862-8652, Japan.
Department of Bioscience, School of Agriculture, Tokai University, 9-1-1 Toroku, Higashi-ku, Kumamoto-shi, Kumamoto 862-8652, Japan.
Int J Biol Macromol. 2020 Dec 1;164:3259-3267. doi: 10.1016/j.ijbiomac.2020.08.197. Epub 2020 Aug 28.
The FMN-dependent NADH-indigo reductase gene from the thermophilic bacterium Bacillus smithii was overexpressed in Escherichia coli. The expressed enzyme functioned as a highly thermostable indigo reductase that retained complete activity even after incubation at 100 °C for 10 min. Furthermore, B. smithii indigo reductase exhibited high stability over a wider pH range and longer storage periods compared with indigo reductases previously identified from other sources. The enzyme catalyzed the reduction of various azo compounds and indigo carmine. The crystal structures of the wild-type enzyme in complex with FMN/N-cyclohexyl-2-aminoethanesulfonate (CHES) and the Y151F mutant enzyme in complex with FMN were determined by the molecular replacement method and refined at resolutions of 1.97 and 1.95 Å, respectively. Then, indigo carmine molecule was modeled into the active site using the molecular docking simulation and the binding mode of indigo carmine was elucidated. In addition, the structure of B. cohnii indigo reductase, which is relatively less stable than B. smithii indigo reductase, was constructed by homology modeling. The factor contributing to the considerably higher thermostability of B. smithii indigo reductase was analyzed by comparing its structure with that of B. cohnii indigo reductase, which revealed that intersubunit aromatic interactions (F105-F172' and F172-F105') may be responsible for the high thermostability of B. smithii indigo reductase. Notably, site-directed mutagenesis results showed that F105 plays a major role in the intersubunit aromatic interaction.
嗜热细菌史密斯芽孢杆菌中依赖黄素单核苷酸（FMN）的NADH-靛蓝还原酶基因在大肠杆菌中过表达。所表达的酶作为一种高度耐热的靛蓝还原酶，即使在100℃孵育10分钟后仍保留完全活性。此外，与先前从其他来源鉴定的靛蓝还原酶相比，史密斯芽孢杆菌靛蓝还原酶在更宽的pH范围内和更长的储存期内表现出高稳定性。该酶催化各种偶氮化合物和靛蓝胭脂红的还原反应。通过分子置换法测定了与FMN/N-环己基-2-氨基乙磺酸盐（CHES）复合的野生型酶以及与FMN复合的Y151F突变型酶的晶体结构，分辨率分别为1.97 Å和1.95 Å，并进行了优化。然后，使用分子对接模拟将靛蓝胭脂红分子模拟到活性位点，并阐明了靛蓝胭脂红的结合模式。此外，通过同源建模构建了比史密斯芽孢杆菌靛蓝还原酶稳定性相对较低的科恩芽孢杆菌靛蓝还原酶的结构。通过将史密斯芽孢杆菌靛蓝还原酶的结构与科恩芽孢杆菌靛蓝还原酶的结构进行比较，分析了导致史密斯芽孢杆菌靛蓝还原酶具有显著更高热稳定性的因素，结果表明亚基间芳香族相互作用（F105-F172'和F172-F105'）可能是史密斯芽孢杆菌靛蓝还原酶高热稳定性的原因。值得注意的是，定点诱变结果表明F105在亚基间芳香族相互作用中起主要作用。
