Molecular mechanism of regulation of the purine salvage enzyme XPRT by the alarmones pppGpp, ppGpp, and pGpp

Abstract

1The alarmones pppGpp and ppGpp mediate starvation response and maintain purine homeostasis 2 to protect bacterial species. Xanthine phosphoribosyltransferase (XPRT) is a purine salvage 3 enzyme that produces the nucleotide XMP from PRPP and xanthine. Combining structural, 4 biochemical and genetic analyses, we show that pppGpp and ppGpp, as well as a third putative 5 alarmone pGpp, all directly interact with XPRT and inhibit XPRT activity by competing with its 6 substrate PRPP. Structural analysis reveals that ppGpp binds the PRPP binding motif within the 7 XPRT active site. This motif is present in another (p)ppGpp target, the purine salvage enzyme 8HPRT, suggesting evolutionary conservation in different enzymes. However, XPRT oligomeric 9 interaction is distinct from HPRT in that XPRT forms a symmetric dimer with two (p)ppGpp 10 binding sites at the dimer interface. This results in two distinct regulatory features. First, XPRT 11 cooperatively binds (p)ppGpp with a Hill coefficient of 2. Also, XPRT displays differential 12 regulation by the alarmones as it is potently inhibited by both ppGpp and pGpp, but only 13 modestly by pppGpp. Lastly, we demonstrate that the alarmones are necessary for protecting 14 GTP homeostasis against excess environmental xanthine in Bacillus subtilis, suggesting that 15 regulation of XPRT is key for regulating the purine salvage pathway. 16 PRPP share the same pocket, and the 3′-phosphates of ppGpp overlap with the 1′-phosphates of 132 PRPP ( Figure 5B). 133Next, we examined whether (p)ppGpp competes with PRPP to inhibit XPRT using 134 steady-state kinetics. We measured initial velocities of XPRT enzymatic reaction by examining 135 the rate of synthesis of XMP at varied pppGpp and PRPP concentrations ( Figure 5C). The data 136 best fit a global competitive inhibition model, demonstrating that (p)ppGpp competes with PRPP 137 ( Figure 5C and 5D). In addition, the kinetic data yielded a Ki for pppGpp of 2.5 μM and a Km for 138

The nucleotide pGpp acts as a third alarmone in Bacillus, with functions distinct from those of (p)ppGpp

“…These results confirmed strong pGpp binding to guanosine nucleotide synthesis proteins (Hpt1, Gmk, and Xpt). Enzyme kinetic assay of Xpt 32 and HprT (Fig. 3e ) confirmed that pGpp inhibited their activities more potently than ppGpp and pppGpp.…”

The stringent response and physiological roles of (pp)pGpp in bacteria

“…Both HprT and XprT use PRPP as a phosphoribose donor to convert the purine bases hypoxanthine and guanine to IMP and GMP (HprT) or xanthine to XMP (XprT) (FIG. 2a) 4,64,88,96 . XprT is most potently inhibited by pGpp and ppGpp, which bind at the PRPP binding site (FIG.…”

“…XprT is most potently inhibited by pGpp and ppGpp, which bind at the PRPP binding site (FIG. 2b) and protect cells from excess environmental xanthine 96 . The (pp)pGpp-bound form of HprT exists as an apo-tetramer (FIG.…”

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 5′-monophosphate 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.…”