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The significance of EXDD and RXKD motif conservation in Rel proteins.

Sajish M, Kalayil S, Verma SK, Nandicoori VK, Prakash B - J. Biol. Chem. (2009)

Bottom Line: 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'P(alpha)-O-P(beta) bond of GTP/GDP or (p)ppGpp.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur 208016, India.

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 Mg(2+) 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'P(alpha)-O-P(beta) 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.

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The new catalytic center involves Pα-O-Pβ bond cleavage of GTP/GDP, (p)ppGpp, and is independent of the pyrophosphoryl transfer reaction from ATP. MT-RelM. tb was used in (p)ppGpp synthesis reactions with [γ-32P]GTP, GTP, and AMP-CPP, a non-hydrolysable ATP analog were used in lane 2. Lanes 1 and 3 contain only [γ-32P]GTP and only [γ-32P]ATP, respectively. The pyrophosphate (PPi) released due to Pα-O-Pβ bond cleavage is indicated in dotted circle.
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fig5: The new catalytic center involves Pα-O-Pβ bond cleavage of GTP/GDP, (p)ppGpp, and is independent of the pyrophosphoryl transfer reaction from ATP. MT-RelM. tb was used in (p)ppGpp synthesis reactions with [γ-32P]GTP, GTP, and AMP-CPP, a non-hydrolysable ATP analog were used in lane 2. Lanes 1 and 3 contain only [γ-32P]GTP and only [γ-32P]ATP, respectively. The pyrophosphate (PPi) released due to Pα-O-Pβ bond cleavage is indicated in dotted circle.

Mentions: To verify the possibility that pGpp is formed due to a hydrolysis of the Pα-O-Pβ bond of GTP/GDP, GMPCPP, a GTP analogue with a carbon in place of an oxygen between Pα and Pβ was used to hinder the aforesaid ester hydrolysis (lane 7, Fig. 4A). Indeed, lane 7 shows the synthesis of pp(c)pGpp, but not pGpp, emphasizing the need for a hydrolysable oxygen between Pα and Pβ of GTP/GDP to synthesize pGpp. Hence, pGpp was synthesized due to the aforesaid novel catalytic center, which was further substantiated by the release of a pyrophosphate from [γ-32P]GTP (Fig. 5), as discussed below. It appears that introducing EXDD resulted in the formation of a new catalytic site to synthesize pGpp by either hydrolyzing GTP/GDP to GMP, which then accepts a pyrophosphate from ATP, or by hydrolyzing (p)ppGpp to pGpp. A critical role for the C-terminal region in catalyzing this reaction was brought out by the fact that only the full-length protein (carrying EXDD mutation) can synthesize pGpp. Therefore, the reaction catalyzed by full-length WT-RelAE. coli, possessing an EXDD, was also examined for the presence of pGpp. The autoradiogram in Fig. 4D, showing a spot at an identical position (lane 2), confirmed that it also synthesized pGpp, although the amounts are insignificant compared with the amount of (p)ppGpp. As anticipated, pGpp synthesis was abolished by MT-RelAE. coli, with EXDD → RXKD substitution (lane 3, Fig. 4D), reiterating the need of EXDD for this activity. Further experiments were designed to understand this novel catalytic subsite.


The significance of EXDD and RXKD motif conservation in Rel proteins.

Sajish M, Kalayil S, Verma SK, Nandicoori VK, Prakash B - J. Biol. Chem. (2009)

The new catalytic center involves Pα-O-Pβ bond cleavage of GTP/GDP, (p)ppGpp, and is independent of the pyrophosphoryl transfer reaction from ATP. MT-RelM. tb was used in (p)ppGpp synthesis reactions with [γ-32P]GTP, GTP, and AMP-CPP, a non-hydrolysable ATP analog were used in lane 2. Lanes 1 and 3 contain only [γ-32P]GTP and only [γ-32P]ATP, respectively. The pyrophosphate (PPi) released due to Pα-O-Pβ bond cleavage is indicated in dotted circle.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2666561&req=5

fig5: The new catalytic center involves Pα-O-Pβ bond cleavage of GTP/GDP, (p)ppGpp, and is independent of the pyrophosphoryl transfer reaction from ATP. MT-RelM. tb was used in (p)ppGpp synthesis reactions with [γ-32P]GTP, GTP, and AMP-CPP, a non-hydrolysable ATP analog were used in lane 2. Lanes 1 and 3 contain only [γ-32P]GTP and only [γ-32P]ATP, respectively. The pyrophosphate (PPi) released due to Pα-O-Pβ bond cleavage is indicated in dotted circle.
Mentions: To verify the possibility that pGpp is formed due to a hydrolysis of the Pα-O-Pβ bond of GTP/GDP, GMPCPP, a GTP analogue with a carbon in place of an oxygen between Pα and Pβ was used to hinder the aforesaid ester hydrolysis (lane 7, Fig. 4A). Indeed, lane 7 shows the synthesis of pp(c)pGpp, but not pGpp, emphasizing the need for a hydrolysable oxygen between Pα and Pβ of GTP/GDP to synthesize pGpp. Hence, pGpp was synthesized due to the aforesaid novel catalytic center, which was further substantiated by the release of a pyrophosphate from [γ-32P]GTP (Fig. 5), as discussed below. It appears that introducing EXDD resulted in the formation of a new catalytic site to synthesize pGpp by either hydrolyzing GTP/GDP to GMP, which then accepts a pyrophosphate from ATP, or by hydrolyzing (p)ppGpp to pGpp. A critical role for the C-terminal region in catalyzing this reaction was brought out by the fact that only the full-length protein (carrying EXDD mutation) can synthesize pGpp. Therefore, the reaction catalyzed by full-length WT-RelAE. coli, possessing an EXDD, was also examined for the presence of pGpp. The autoradiogram in Fig. 4D, showing a spot at an identical position (lane 2), confirmed that it also synthesized pGpp, although the amounts are insignificant compared with the amount of (p)ppGpp. As anticipated, pGpp synthesis was abolished by MT-RelAE. coli, with EXDD → RXKD substitution (lane 3, Fig. 4D), reiterating the need of EXDD for this activity. Further experiments were designed to understand this novel catalytic subsite.

Bottom Line: 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'P(alpha)-O-P(beta) bond of GTP/GDP or (p)ppGpp.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur 208016, India.

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 Mg(2+) 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'P(alpha)-O-P(beta) 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.

Show MeSH
Related in: MedlinePlus