Inhibitors of a Na+-pumping NADH-ubiquinone oxidoreductase play multiple roles to block enzyme function
Inhibitors of a Na+-pumping NADH-ubiquinone oxidoreductase play multiple roles to block enzyme function
复制标题
DOI：
10.1074/jbc.ra120.014229
复制
发表时间：
2020-09-04
影响因子：
4.8
通讯作者：
Miyoshi, Hideto
中科院分区：
文献类型：
--
作者：
Masuya, Takahiro;Sano, Yuki;Miyoshi, Hideto
Na+泵辅酶Q-NADH氧化还原酶（Na+-NQR）存在于许多病原菌的呼吸链中，被认为是一种有前途的抗生素靶标。虽然Na+-NQR结构和功能的许多细节是已知的，但有效抑制剂的作用机制还没有很好地理解;阐明这些机制不仅可以推进药物设计策略，还可以提供从核黄素到UQ的末端电子转移的见解。为此，我们使用两种已知抑制剂aurachin和korormicin的光反应衍生物对分离的霍乱弧菌Na（+）-NQR进行了光亲和标记实验。这些抑制剂标记了NqrB亚基的胞质表面结构域，包括突出的N-末端延伸，这可能对调节相邻NqrA亚基中的UQ反应至关重要。标记被短链UQs如泛醌-2阻断。这些抑制剂的光不稳定基团（2-芳基-5-羧基四唑（ACT））与亲核氨基酸反应，因此我们测试了NqrB标记区域中亲核残基的突变，如Asp（49）和Asp（52）（至Ala），并观察到标记产率的适度降低，表明这些残基参与了与ACT的相互作用。我们的结论是，抑制剂干扰UQ反应在两个方面：第一个是阻止NqrA和NqrB之间的细胞质界面的结构重排，第二个是UQ结合在这个界面区域的直接障碍。不寻常的竞争行为之间的光反应抑制剂和各种竞争对手证实了我们以前的命题，可能有两个抑制剂结合位点的Na+-NQR。
The Na+-pumping NADH-ubiquinone (UQ) oxidoreductase (Na+-NQR) is present in the respiratory chain of many pathogenic bacteria and is thought to be a promising antibiotic target. Whereas many details of Na+-NQR structure and function are known, the mechanisms of action of potent inhibitors is not well-understood; elucidating the mechanisms would not only advance drug design strategies but might also provide insights on a terminal electron transfer from riboflavin to UQ. To this end, we performed photoaffinity labeling experiments using photoreactive derivatives of two known inhibitors, aurachin and korormicin, on isolatedVibrio choleraeNa(+)-NQR. The inhibitors labeled the cytoplasmic surface domain of the NqrB subunit including a protruding N-terminal stretch, which may be critical to regulate the UQ reaction in the adjacent NqrA subunit. The labeling was blocked by short-chain UQs such as ubiquinone-2. The photolabile group (2-aryl-5-carboxytetrazole (ACT)) of these inhibitors reacts with nucleophilic amino acids, so we tested mutations of nucleophilic residues in the labeled region of NqrB, such as Asp(49)and Asp(52)(to Ala), and observed moderate decreases in labeling yields, suggesting that these residues are involved in the interaction with ACT. We conclude that the inhibitors interfere with the UQ reaction in two ways: the first is blocking structural rearrangements at the cytoplasmic interface between NqrA and NqrB, and the second is the direct obstruction of UQ binding at this interfacial area. Unusual competitive behavior between the photoreactive inhibitors and various competitors corroborates our previous proposition that there may be two inhibitor binding sites in Na+-NQR.