Department of Bacteriology, University of Wisconsin-Madison, 1550 Linden Dr., Madison, WI 53706, USA.
Department of Biomolecular Chemistry, University of Wisconsin-Madison, 440 Henry Mall, Madison, WI 53706, USA.
J Mol Biol. 2020 Jun 26;432(14):4108-4126. doi: 10.1016/j.jmb.2020.05.013. Epub 2020 May 21.
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.
pppGpp 和 ppGpp 作为警报素，可介导细菌的饥饿反应和嘌呤稳态维持，从而起到保护细菌的作用。在厚壁菌门和拟杆菌门的细菌中，黄嘌呤磷酸核糖基转移酶（XPRT）是一种嘌呤补救酶，可将 PRPP 和黄嘌呤转化为核苷酸 XMP。本研究结合结构、生化和遗传分析表明，pppGpp 和 ppGpp 以及第三种新鉴定的警报素 pGpp 均可与革兰氏阳性菌枯草芽孢杆菌的 XPRT 直接相互作用，并通过与底物 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。该基序存在于另一种（p）ppGpp 靶标嘌呤补救酶 HPRT 中，提示不同酶之间存在进化保守性。然而，XPRT 的寡聚相互作用不同于 HPRT，因为 XPRT 以对称二聚体的形式存在，两个（p）ppGpp 结合位点位于二聚体界面上。（p）ppGpp 与界面上的 XPRT 桥接环相互作用，导致 XPRT 协同结合（p）ppGpp。此外，XPRT 受到警报素的差异化调节，因为它受到 ppGpp 和 pGpp 的强烈抑制，但仅受到 pppGpp 的适度抑制。最后，我们证明警报素对于保护枯草芽孢杆菌中的 GTP 稳态免受环境中过量黄嘌呤的影响是必需的，这表明 XPRT 的调节对于嘌呤补救途径的调节至关重要。
