RESUMO
In response to starvation, virtually all bacteria pyrophosphorylate the 3'-hydroxy group of GTP or GDP to produce two messenger nucleotides collectively denoted as (p)ppGpp. Also known as alarmones, (p)ppGpp reprograms bacterial physiology to arrest growth and promote survival. Intriguingly, although cellular concentration of dGTP is two orders of magnitude lower than that of GTP, alarmone synthetases are highly selective against using 2'-deoxyguanosine (2dG) nucleotides as substrates. We thus hypothesize that production of 2dG alarmone, (p)pp(dG)pp, is highly deleterious, which drives a strong negative selection to exclude 2dG nucleotides from alarmone signaling. In this work, we show that the B. subtilis SasB synthetase prefers GDP over dGDP with 65,000-fold higher kcat/Km, a specificity stricter than RNA polymerase selecting against 2'-deoxynucleotides. Using comparative chemical proteomics, we found that although most known alarmone-binding proteins in Escherichia coli cannot distinguish ppGpp from pp(dG)pp, hydrolysis of pp(dG)pp by the essential hydrolase, SpoT, is 1,000-fold slower. This inability to degrade 2'-deoxy-3'-pyrophosphorylated substrate is a common feature of the alarmone hydrolase family. We further show that SpoT is a binuclear metallopyrophoshohydrolase and that hydrolysis of ppGpp and pp(dG)pp shares the same metal dependence. Our results support a model in which 2'-OH directly coordinates the Mn2+ at SpoT active center to stabilize the hydrolysis-productive conformation of ppGpp. Taken together, our study reveals a vital role of 2'-OH in alarmone degradation, provides new insight on the catalytic mechanism of alarmone hydrolases, and leads to the conclusion that 2dG nucleotides must be strictly excluded from alarmone synthesis because bacteria lack the key machinery to down-regulate such products.
RESUMO
Bacteria produce a variety of nucleotide second messengers to adapt to their surroundings. Although chemically similar, the nucleotides guanosine penta- and tetraphosphate [(p)ppGpp] and adenosine penta- and tetraphosphate [(p)ppApp] have distinct functions in bacteria. (p)ppGpp mediates survival under nutrient-limiting conditions and its intracellular levels are regulated by synthetases and hydrolases belonging to the RelA-SpoT homolog (RSH) family of enzymes. By contrast, (p)ppApp is not known to be involved in nutrient stress responses and is synthesized by RSH-resembling toxins that inhibit the growth of bacterial cells. However, it remains unclear whether there exists a family of hydrolases that specifically act on (p)ppApp to reverse its toxic effects. Here, we present the structure and biochemical characterization of adenosine 3'-pyrophosphohydrolase 1 (Aph1), the founding member of a monofunctional (p)ppApp hydrolase family of enzymes. Our work reveals that Aph1 adopts a histidine-aspartate (HD)-domain fold characteristic of phosphohydrolase metalloenzymes and its activity mitigates the growth inhibitory effects of (p)ppApp-synthesizing toxins. Using an informatic approach, we identify over 2,000 putative (p)ppApp hydrolases that are widely distributed across bacterial phyla and found in diverse genomic contexts, and we demonstrate that 12 representative members hydrolyze ppApp. In addition, our in silico analyses reveal a unique molecular signature that is specific to (p)ppApp hydrolases, and we show that mutation of two residues within this signature broadens the specificity of Aph1 to promiscuously hydrolyze (p)ppGpp in vitro. Overall, our findings indicate that like (p)ppGpp hydrolases, (p)ppApp hydrolases are widespread in bacteria and may play important and underappreciated role(s) in bacterial physiology.