Qi Yaning, Rao Feng, Luo Zhen, Liang Zhao-Xun
Division of Chemical Biology and Biotechnology, School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551.
Biochemistry. 2009 Nov 3;48(43):10275-85. doi: 10.1021/bi901121w.
The cytoplasmic protein AxDGC2 regulates cellulose synthesis in the obligate aerobe Acetobacter xylinum by controlling the cellular concentration of the cyclic dinucleotide messenger c-di-GMP. AxDGC2 contains a Per-Arnt-Sim (PAS) domain and two putative catalytic domains (GGDEF and EAL) for c-di-GMP metabolism. We found that the PAS domain of AxDGC2 binds a flavin adenine dinucleotide (FAD) cofactor noncovalently. The redox status of the FAD cofactor modulates the catalytic activity of the GGDEF domain for c-di-GMP synthesis, with the oxidized form exhibiting higher catalytic activity and stronger substrate inhibition. The results suggest that AxDGC2 is a signaling protein that regulates the cellular c-di-GMP level in response to the change in cellular redox status or oxygen concentration. Moreover, several residues predicated to be involved in FAD binding and signal transduction were mutated to examine the impact on redox potential and catalytic activity. Despite the minor perturbation of redox potential and unexpected modification of FAD in one of the mutants, none of the single mutations was able to completely disrupt the transmission of the signal to the GGDEF domain, indicating that the change in the FAD redox state can still trigger structural changes in the PAS domain probably by using substituted hydrogen-bonded water networks. Meanwhile, although the EAL domain of AxDGC2 was found to be catalytically inactive toward c-di-GMP, it was capable of hydrolyzing some phosphodiester bond-containing nonphysiological substrates. Together with the previously reported oxygen-dependent activity of the homologous AxPDEA1, the results provided new insight into relationships among oxygen level, c-di-GMP concentration, and cellulose synthesis in A. xylinum.
细胞质蛋白AxDGC2通过控制环二核苷酸信使c-di-GMP的细胞浓度来调节专性需氧菌木醋杆菌中的纤维素合成。AxDGC2包含一个Per-Arnt-Sim（PAS）结构域和两个用于c-di-GMP代谢的假定催化结构域（GGDEF和EAL）。我们发现AxDGC2的PAS结构域非共价结合黄素腺嘌呤二核苷酸（FAD）辅因子。FAD辅因子的氧化还原状态调节GGDEF结构域对c-di-GMP合成的催化活性，氧化形式表现出更高的催化活性和更强的底物抑制作用。结果表明，AxDGC2是一种信号蛋白，可响应细胞氧化还原状态或氧气浓度的变化来调节细胞c-di-GMP水平。此外，预测参与FAD结合和信号转导的几个残基发生了突变，以研究对氧化还原电位和催化活性的影响。尽管其中一个突变体的氧化还原电位受到轻微扰动且FAD发生意外修饰，但没有一个单突变能够完全破坏信号向GGDEF结构域的传递，这表明FAD氧化还原状态的变化可能仍通过使用取代的氢键水网络触发PAS结构域的结构变化。同时，尽管发现AxDGC2的EAL结构域对c-di-GMP无催化活性，但它能够水解一些含磷酸二酯键的非生理性底物。与先前报道的同源AxPDEA1的氧依赖性活性一起，这些结果为木醋杆菌中氧水平、c-di-GMP浓度和纤维素合成之间的关系提供了新的见解。
