Molecular Mechanism of Regulation of the Purine Salvage Enzyme XPRT by the Alarmones pppGpp, ppGpp, and pGpp

Abstract

The alarmones pppGpp and ppGpp mediate starvation response and maintain purine homeostasis to protect bacterial species. Xanthine phosphoribosyltransferase (XPRT) is a purine salvage 3 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 putative alarmone pGpp, all directly interact with XPRT 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. This results in two distinct regulatory features. First, XPRT cooperatively binds (p)ppGpp with a Hill coefficient of 2. 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 Bacillus subtilis, suggesting that regulation of XPRT is key for regulating the purine salvage pathway.

The nucleotide messenger (p)ppGpp is a co-repressor of the purine synthesis transcription regulator PurR in Firmicutes

“…In 383 one case, (p)ppGpp binds to many GTPases through their GTP-binding motif (Buglino et al, 2002a;Fan et al, 2015;Kihira et al, 2012;Pausch et al, 2018). In another case, (p)ppGpp binds to a conserved PRPP-binding motif in the PRT fold in two enzymatic targets, the purine salvage enzymes HPRT and XPRT (Anderson et al, 2019(Anderson et al, , 2020. The (p)ppGpp binding site on PurR is the third PRT site also found to bind (p)ppGpp ( Figure 6A).…”

“…(p)ppGpp has been shown to bind to the RNA polymerase core enzyme in Proteobacteria (Molodtsov et al, 2018;Ross et al, 2013Ross et al, , 2016. In contrast, in Firmicutes (p)ppGpp regulates many targets, including nucleotide synthesis enzymes, ribosome associated GTPases, and DNA replication enzymes (Anderson et al, 2019(Anderson et al, , 2020Corrigan et al, 2016;Kriel et al, 2012;Wang et al, 2007;Yang et al, 2020), but has not been shown to regulate transcription directly (Liu et al, 2015a). To uncover potential (p)ppGpp regulated transcriptional targets, we conducted a proteome based screen for proteins that directly bind (p)ppGpp in the pathogenic firmicute Bacillus anthracis (Yang et al, 2020).…”

“…In XPRT, the (p)ppGpp binding sites are cooperatively linked across a monomer-monomer interface. (p)ppGpp binding creates an electrostatic network with a bridge loop of the other monomer, inducing XPRT dimerization (Anderson et al, 2020). The PurR-ppGpp interaction introduces allosteric change of a transcription factor as a third allosteric property associated with the (p)ppGpp interacting with the same motif.…”

“…Perhaps most intriguingly, during the evolutionary co-opting of the PRT enzyme as the PurR transcription regulatory domain, not only is (p)ppGpp binding conserved, so is the (p)ppGpp-PRPP competition ( Figure 6A). PRPP is the common substrates for HPRT and XPRT, and (p)ppGpp inhibits HPRT and XPRT by competing with PRPP (Anderson et al, 2019(Anderson et al, , 2020).…”

Structural Analysis of (p)ppGpp Reveals Its Versatile Binding Pattern for Diverse Types of Target Proteins

“…There are six crystal structures of (p)ppGpp and nucleotide metabolic enzymes complexes available in the Protein Data Bank which includes Xanthine phosphoribosyltransferase (XPRT) (Anderson et al, 2020), Pyrimidine/purine nucleotide 5monophosphate nucleosidase (nucleosidase, PpnN) (Zhang et al, 2019), Hypoxanthine phosphoribosyltransferase (HPRT) (Anderson et al, 2019), Amidophosphoribosyltransferase (PurF) (Wang et al, 2019), Guanylate kinase (Liu et al, 2015; Table 1). A most striking feature of the structures of these enzyme-(p)ppGpp complexes are oligomeric stoichiometry in these complexes.…”