From the Department of Molecular Biology and Genetics, Centre for Bacterial Stress Response and Persistence, Gustav Wieds Vej 10c, DK-8000 Aarhus C, Denmark.
the University of Tartu, Institute of Technology, Nooruse 1, 50411 Tartu, Estonia.
J Biol Chem. 2018 Mar 2;293(9):3254-3264. doi: 10.1074/jbc.RA117.001374. Epub 2018 Jan 11.
The stringent response is a global reprogramming of bacterial physiology that renders cells more tolerant to antibiotics and induces virulence gene expression in pathogens in response to stress. This process is driven by accumulation of the intracellular alarmone guanosine-5'-di(tri)phosphate-3'-diphosphate ((p)ppGpp), which is produced by enzymes of the RelA SpoT homologue (RSH) family. The Gram-positive Firmicute pathogen, , encodes three RSH enzymes: a multidomain RSH (Rel) that senses amino acid starvation on the ribosome and two small alarmone synthetase (SAS) enzymes, RelQ (SAS1) and RelP (SAS2). In , RelQ (SAS1) was shown to form a tetramer that is activated by pppGpp and inhibited by single-stranded RNA, but the structural and functional regulation of RelP (SAS2) is unexplored. Here, we present crystal structures of RelP in two major functional states, pre-catalytic (bound to GTP and the non-hydrolyzable ATP analogue, adenosine 5'-(α,β-methylene)triphosphate (AMP-CPP)), and post-catalytic (bound to pppGpp). We observed that RelP also forms a tetramer, but unlike RelQ (SAS1), it is strongly inhibited by both pppGpp and ppGpp and is insensitive to inhibition by RNA. We also identified putative metal ion-binding sites at the subunit interfaces that were consistent with the observed activation of the enzyme by Zn ions. The structures reported here reveal the details of the catalytic mechanism of SAS enzymes and provide a molecular basis for understanding differential regulation of SAS enzymes in Firmicute bacteria.
stringent response 是一种全局性的细菌生理重编程，可使细胞对抗生素更具耐受性，并在病原体中诱导毒力基因表达以应对应激。这一过程是由细胞内警报核苷酸鸟苷-5'-二（三）磷酸-3'-二磷酸 ((p)ppGpp) 的积累所驱动的，该物质由 RelA SpoT 同源物 (RSH) 家族的酶产生。革兰氏阳性厚壁菌病原体 ， 编码三种 RSH 酶：一种多结构域 RSH (Rel)，可在核糖体上感应氨基酸饥饿，以及两种小警报核苷酸合成酶 (SAS) 酶，RelQ (SAS1) 和 RelP (SAS2)。在 中，RelQ (SAS1) 被证明形成四聚体，该四聚体可被 pppGpp 激活，被单链 RNA 抑制，但 RelP (SAS2) 的结构和功能调节尚未被探索。在这里，我们展示了 RelP 在两种主要功能状态下的晶体结构，即预催化态（与 GTP 和非水解型 ATP 类似物，腺苷 5'-(α,β-亚甲基)三磷酸 (AMP-CPP) 结合）和后催化态（与 pppGpp 结合）。我们观察到 RelP 也形成四聚体，但与 RelQ (SAS1) 不同，它被 pppGpp 和 ppGpp 强烈抑制，并且对 RNA 的抑制不敏感。我们还在亚基界面上确定了潜在的金属离子结合位点，这些位点与观察到的酶被 Zn 离子激活一致。这里报道的结构揭示了 SAS 酶的催化机制的细节，并为理解厚壁菌中 SAS 酶的差异调节提供了分子基础。
