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Molecular Mechanism of Regulation of the Purine Salvage Enzyme XPRT by the Alarmones pppGpp, ppGpp, and pGpp.
Journal of Molecular Biology ( IF 5.6 ) Pub Date : 2020-05-21 , DOI: 10.1016/j.jmb.2020.05.013
Brent W Anderson 1 , Aili Hao 1 , Kenneth A Satyshur 2 , James L Keck 2 , Jue D Wang 1
Affiliation  

The alarmones pppGpp and ppGpp mediate starvation response and maintain purine homeostasis to protect bacteria. In the bacterial phyla Firmicutes and Bacteroidetes, xanthine phosphoribosyltransferase (XPRT) is a purine salvage enzyme that produces the nucleotide XMP from PRPP and xanthine. Combining structural, biochemical, and genetic analyses, we show that pppGpp and ppGpp, as well as a third newly identified alarmone pGpp, all directly interact with XPRT from the Gram-positive bacterium Bacillus subtilis and inhibit XPRT activity by competing with its substrate PRPP. Structural analysis reveals that ppGpp binds the PRPP binding motif within the XPRT active site. This motif is present in another (p)ppGpp target, the purine salvage enzyme HPRT, suggesting evolutionary conservation in different enzymes. However, XPRT oligomeric interaction is distinct from HPRT in that XPRT forms a symmetric dimer with two (p)ppGpp binding sites at the dimer interface. (p)ppGpp's interaction with an XPRT bridging loop across the interface results in XPRT cooperatively binding (p)ppGpp. Also, XPRT displays differential regulation by the alarmones as it is potently inhibited by both ppGpp and pGpp, but only modestly by pppGpp. Lastly, we demonstrate that the alarmones are necessary for protecting GTP homeostasis against excess environmental xanthine in B. subtilis, suggesting that regulation of XPRT is key for regulating the purine salvage pathway.



中文翻译:

警报蛋白pppGpp,ppGpp和pGpp调节嘌呤清除酶XPRT的分子机理。

警铃pppGpp和ppGpp介导饥饿反应并维持嘌呤稳态以保护细菌。在细菌门扇菌属和拟杆菌属中,黄嘌呤磷酸核糖基转移酶(XPRT)是一种嘌呤挽救酶,可从PRPP和黄嘌呤产生核苷酸XMP。结合结构,生化和遗传分析,我们显示pppGpp和ppGpp以及第三个新发现的警报蛋白pGpp都直接与革兰氏阳性细菌枯草芽孢杆菌的XPRT相互作用通过与底物PRPP竞争来抑制XPRT活性。结构分析表明ppGpp结合XPRT活性位点内的PRPP结合基序。该基序存在于另一个(p)ppGpp靶标中,嘌呤挽救酶HPRT,表明在不同酶中的进化保守性。但是,XPRT低聚物的相互作用不同于HPRT,因为XPRT形成对称的二聚体,在二聚体界面处有两个(p)ppGpp结合位点。(p)ppGpp与跨接口的XPRT桥接环的交互导致XPRT协作绑定(p)ppGpp。同样,XPRT显示出警报物的微分调节,因为它被ppGpp和pGpp均有效抑制,但仅被pppGpp适度抑制。最后,我们证明了报警器对于保护GTP稳态免受过量环境黄嘌呤的侵害是必要的。枯草芽孢杆菌,表明XPRT的调节是调节嘌呤挽救途径的关键。

更新日期:2020-06-23
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