Cao Xinhua, Di Mingxiao, Wang Jun
Key Laboratory of Aquatic Botany and Watershed Ecology, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan, 430074, China; University of Chinese Academy of Sciences, Beijing, 100049, China.
Key Laboratory of Aquatic Botany and Watershed Ecology, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan, 430074, China; Sino-Africa Joint Research Center, Chinese Academy of Sciences, Wuhan, 430074, China.
J Mol Graph Model. 2017 Nov;78:213-220. doi: 10.1016/j.jmgm.2017.10.020. Epub 2017 Oct 31.
Azoreductase from Shewanella oneidensis MR-1 (soAzoR) possesses great potential in cleaving azo bond of azo dyes during degradation progress. However, detailed information on interaction of soAzoR with either prosthetic group or substrate remains unavailable, mainly due to the absence of crystallization of soAzoR. In order to unravel these mechanisms, we firstly built the tertiary structure of soAzoR and then computationally predicted the binding mode of FMN, NADH and a model dye, methyl red (MR). Ten residues of soAzoR, which are predicted to participate in ligands binding, were separately substituted for either alanine or phenylalanine to confirm their function. The homologous modeling result reveals soAzoR employs a typical Rossmann fold. In terms of ligand binding modes, the isoalloxazine ring of FMN is stabilized in planar conformation by amino acids in the loop L6 and L9 region. NADH and MR is superposed against the isoalloxazine ring with an angle and the distance from C4 atom of NADH and azo bond of MR to N5 atom of FMN is 4.3Å and 4.6Å, respectively. The result of predicted interaction and enzyme kinetic analysis suggests that Asn96, Gly140 and Gly141 are crucial for FMN and MR binding; Tyr119 and Phe161 are more meaningful for NADH binding; Ser16 plays an important role in appropriately binding of both FMN and NADH.
来自希瓦氏菌MR-1（soAzoR）的偶氮还原酶在偶氮染料降解过程中具有裂解偶氮键的巨大潜力。然而，关于soAzoR与辅基或底物相互作用的详细信息仍然缺乏，主要原因是soAzoR没有结晶。为了揭示这些机制，我们首先构建了soAzoR的三级结构，然后通过计算预测了FMN、NADH和一种模型染料甲基红（MR）的结合模式。预测参与配体结合的soAzoR的10个残基分别被丙氨酸或苯丙氨酸取代以确认其功能。同源建模结果表明soAzoR采用典型的Rossmann折叠。在配体结合模式方面，FMN的异咯嗪环通过环L6和L9区域的氨基酸稳定在平面构象中。NADH和MR与异咯嗪环呈一定角度叠加，NADH的C4原子和MR的偶氮键到FMN的N⁵原子的距离分别为4.3Å和4.6Å。预测的相互作用结果和酶动力学分析表明，Asn96、Gly140和Gly141对FMN和MR的结合至关重要；Tyr119和Phe161对NADH的结合更有意义；Ser16在FMN和NADH的适当结合中起重要作用。
