Degradation of Guanosine 3'-diphosphate 5'-diphosphate in vitro by the spoT Gene Product of Escherichia coli

Abstract

Guanosine 3'-diphosphate 5'-diphosphate (ppGpp) is rapidly degraded to guanosine 5'-diphosphate (ppG) and probably pyrophosphate by an enzyme present in the ribosomal fraction prepared from spoT' strains of Escherichia coli. The ppGpp-degrading enzyme was released from the ribosomes during dissociation at low ionic strength. Ribosomes are not essential for degradation of ppGpp, and decay of ppGpp is strictly dependent on manganese ions. The reaction is sensitive to inhibition by tetracycline, which can be reversed by MnC12, indicating that the inhibitory effect is due to the previously described chelating properties of the antibiotic. When the ppGpp-degrading enzyme was complemented with adenosine 5'-triphosphate (pppA) and a nucleoside diphosphate kinase, decay of ppGpp was accelerated yielding pppG and ppG as major products. In the absence of pppA we have been unable to detect the ppGpp-degrading enzyme in various SPOT-mutant strains indicating that this enzyme is the spoT gene product.

Mechanism of the in vitro breakdown of guanosine 5'-diphosphate 3'-diphosphate in Escherichia coli.

“…In the presence of pppA, the decay reaction is significantly stimulated and the 3'-pyrophosphoryl group of ppGpp exchanges, displacing the My and ,3 phosphates of pppA. The former reaction is due to a nucleoside diphosphate kinase-dependent reaction that phosphorylates ppG to pppG (21). This finding is in agreement with in vivo data that the rate constant of ppGpp degradation is decreased by an order of magnitude when production of high-energy-containing compounds is blocked (24,25).…”

Degradation of ppGpp by Nudix Pyrophosphatase Modulates the Transition of Growth Phase in the Bacterium Thermus thermophilus

“…Notably, Ndx8 produced pGp by sequential hydrolysis of two phosphodiester bonds in ppGpp, whereas pGp and two other intermediates could not be detected from the metabolomics screening in this study. For E. coli, previous in vitro studies reported the activity of (p)ppGpp degradation resulting in the production of pGp, ppGp, and pGpp (9), which are regarded as probable sources for the production of guanosine polyphosphate derivatives in vivo (10,55). Moreover, one of such a product, ppGp, is proposed to act as a strong stringent factor inhibiting purine biosynthesis (56,57).…”

Isolation of a pyrophosphorylase from Bacillus subtilis and Bacillus stearothermophilus that specifically degrades guanosine 3′‐diphosphate 5′‐diphosphate

“…Heinemeyer and according to [9]; 32P-labeled ppGpp substrates are designated with an asterisk over the 'p'; protein was estimated as in [9].…”

“…In this study, we have investigated the in vitro degradation of ppGpp in Bacillus stearothermophilus and Bacillus subtilis, in particular we were interested whether ppGpp is degraded by a similar mechanism as has been reported for E. coli [7,8]. In the latter strain a manganese-dependent enzyme has been isolated [9] that released pyrophosphate from the 3'-position of ppGpp yielding ppG [lo].…”

“…32P-labeled pyrophosphate, identified by treatment with yeast inorganic pyrophosphatase (Sigma), was calculated from the percentage of pp["H]Gcp (or pp [ 3H]Gp$) converted to ppG and '*PPi from E. coli depends on divalent cations, in particular on manganese ions [9]. As shown in table 2 the pyrophosphorylase from the two Bacillus strains shows a similar requirement for manganese ions.…”