Minazzato Gabriele, Gasparrini Massimiliano, Heroux Annie, Sernova Natalia V, Rodionov Dmitry A, Cianci Michele, Sorci Leonardo, Raffaelli Nadia
Department of Agricultural, Food and Environmental Sciences, Polytechnic University of Marche, Ancona, Italy.
Elettra - Sincrotrone Trieste S.C.P.A., Basovizza, Italy.
J Struct Biol. 2022 Dec;214(4):107917. doi: 10.1016/j.jsb.2022.107917. Epub 2022 Nov 1.
Nicotinamide-adenine dinucleotide (NAD) is centrally important to metabolic reactions that involve redox chemistry. In bacteria, NAD biosynthesis is controlled by different transcription factors, depending on the species. Among the four regulators identified so far, the protein NadQ is reported to act as a repressor of the de novo NAD biosynthetic pathway in proteobacteria. Using comparative genomics, a systematic reconstruction of NadQ regulons in thousands of fully sequenced bacterial genomes has been performed, confirming that NadQ is present in α-proteobacteria and some β- and γ-proteobacteria, including pathogens like Bordetella pertussis and Neisseria meningitidis, where it likely controls de novo NAD biosynthesis. Through mobility shift assay and mutagenesis, the DNA binding activity of NadQ from Agrobacterium tumefaciens was experimentally validated and determined to be suppressed by ATP. The crystal structures of NadQ in native form and in complex with ATP were determined, indicating that NadQ is a dimer, with each monomer composed of an N-terminal Nudix domain hosting the effector binding site and a C-terminal winged helix-turn-helix domain that binds DNA. Within the dimer, we found one ATP molecule bound, at saturating concentration of the ligand, in keeping with an intrinsic asymmetry of the quaternary structure. Overall, this study provided the basis for depicting a working model of NadQ regulation mechanism.
烟酰胺腺嘌呤二核苷酸（NAD）对于涉及氧化还原化学的代谢反应至关重要。在细菌中，NAD生物合成由不同的转录因子控制，具体取决于细菌种类。在目前已鉴定出的四种调节因子中，据报道蛋白质NadQ在变形菌中作为从头合成NAD生物合成途径的阻遏物发挥作用。利用比较基因组学，对数千个全基因组测序的细菌基因组中的NadQ调控子进行了系统重建，证实NadQ存在于α-变形菌以及一些β-和γ-变形菌中，包括百日咳博德特氏菌和脑膜炎奈瑟氏菌等病原体，在这些病原体中它可能控制从头合成NAD的生物合成。通过迁移率变动分析和诱变实验，验证了根癌土壤杆菌中NadQ的DNA结合活性，并确定其受到ATP的抑制。测定了天然形式以及与ATP结合的NadQ的晶体结构，表明NadQ是一种二聚体，每个单体由一个包含效应物结合位点的N端Nudix结构域和一个结合DNA的C端翼状螺旋-转角-螺旋结构域组成。在二聚体内，我们发现，在配体饱和浓度下，结合了一个ATP分子，这与四级结构的内在不对称性一致。总体而言，本研究为描绘NadQ调控机制的工作模型提供了基础。
