Abstract
Monofunctional and bifunctional classes of Rel proteins catalyze pyrophosphoryl transfer from ATP to 3′-OH of GTP/GDP to synthesize (p)ppGpp, which is essential for normal microbial physiology and survival. Bifunctional proteins additionally catalyze the hydrolysis of (p)ppGpp. We have earlier demonstrated that although both catalyze identical the (p)ppGpp synthesis reaction, they exhibit a differential response to Mg2+ due to a unique charge reversal in the synthesis domain; an RXKD motif in the synthesis domain of bifunctional protein is substituted by an EXDD motif in that of the monofunctional proteins. Here, we show that these motifs also determine substrate specificities (GTP/GDP), cooperativity, and regulation of catalytic activities at the N-terminal region through the C-terminal region. Most importantly, a mutant bifunctional Rel carrying an EXDD instigates a novel catalytic reaction, resulting in the synthesis of pGpp by an independent hydrolysis of the 5′Pα-O-Pβ bond of GTP/GDP or (p)ppGpp. Further experiments with RelA from Escherichia coli wherein EXDD is naturally present also revealed the presence of pGpp, albeit at low levels. This work brings out the biological significance of RXKD/EXDD motif conservation in Rel proteins and reveals an additional catalytic activity for the monofunctional proteins, prompting an extensive investigation for the possible existence and role of pGpp in the biological system.
Highlights
The adaptability to changing environments determines the survival of an organism
In contrast to the earlier reports, we found that monofunctional RelAE. coli utilizes GDP and bifunctional RelM. tb utilizes GTP as the principal pyrophosphate acceptor, and this specificity is determined by the EXDD and RXKD motifs, respectively
When synthesis reactions were carried out using the N-terminal regions of RelAE. coli and mutant RelM. tb (RXKD 3 EXDD) with ATP and GTP as substrates, we were surprised to observe an additional spot in the autoradiograms apart from pppGpp (8)
Summary
A mutant bifunctional Rel carrying an EXDD instigates a novel catalytic reaction, resulting in the synthesis of pGpp by an independent hydrolysis of the 5P␣-O-P bond of GTP/GDP or (p)ppGpp. Further experiments with RelA from Escherichia coli wherein EXDD is naturally present revealed the presence of pGpp, albeit at low levels. As favorable conditions are restored, the stringent response can be reversed by the hydrolysis of (p)ppGpp to GTP/GDP and pyrophosphate (PPi) by the bifunctional Rel proteins. Earlier, using the N-terminal regions of Rel proteins, we had shown that they differ in utilizing Mg2ϩ for (p)ppGpp synthesis and had attributed this difference to a charge reversal in the synthesis domain where an RXKD in the bifunctional protein is substituted to an EXDD in monofunctional proteins (8). The presence of pGpp in this reaction, albeit at low levels, opens the avenue to explore the significance of pGpp versus (p)ppGpp in microbial physiology
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