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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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A, interchanging RXKD and EXDD motifs in Rel proteins affects (p)ppGpp synthesis. (p)ppGpp synthesis was assayed for the indicated wild type and mutant proteins using either GTP or GDP along with [γ-32P]ATP. The activity of MT-RelAE. coli is plotted while considering WT-RelAE. coli activity to be 100%. The activities of WT-RelM. tb and MT-RelM. tb are also plotted similarly. B, intrinsic fluorescence exhibited by WT-RelAE. coli, MT-RelAE. coli, and W260A mutant of WT-RelAE. coli. C, intrinsic fluorescence exhibited by WT-RelM. tb and MT-RelM. tb. Emission spectra were recorded at λex of 280 nm. The inset shows a structure-based sequence comparison of the catalytic loop of RelS. eq, RelM. tb, and RelAE. coli, with the numbers corresponding to that of RelS. eq.
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fig3: A, interchanging RXKD and EXDD motifs in Rel proteins affects (p)ppGpp synthesis. (p)ppGpp synthesis was assayed for the indicated wild type and mutant proteins using either GTP or GDP along with [γ-32P]ATP. The activity of MT-RelAE. coli is plotted while considering WT-RelAE. coli activity to be 100%. The activities of WT-RelM. tb and MT-RelM. tb are also plotted similarly. B, intrinsic fluorescence exhibited by WT-RelAE. coli, MT-RelAE. coli, and W260A mutant of WT-RelAE. coli. C, intrinsic fluorescence exhibited by WT-RelM. tb and MT-RelM. tb. Emission spectra were recorded at λex of 280 nm. The inset shows a structure-based sequence comparison of the catalytic loop of RelS. eq, RelM. tb, and RelAE. coli, with the numbers corresponding to that of RelS. eq.

Mentions: All the values were determined in the absence of cofactors such as mRNA, tRNA, and/or ribosomes or denaturants such as methanol and detergents, unlike the earlier studies (5, 15). Values obtained from three independent experiments were used for calculating the S.D. MT-RelM. tb (EXDD) was not used here, as (p)ppGpp synthesis was reduced by 90% (see Fig. 3A).


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)

A, interchanging RXKD and EXDD motifs in Rel proteins affects (p)ppGpp synthesis. (p)ppGpp synthesis was assayed for the indicated wild type and mutant proteins using either GTP or GDP along with [γ-32P]ATP. The activity of MT-RelAE. coli is plotted while considering WT-RelAE. coli activity to be 100%. The activities of WT-RelM. tb and MT-RelM. tb are also plotted similarly. B, intrinsic fluorescence exhibited by WT-RelAE. coli, MT-RelAE. coli, and W260A mutant of WT-RelAE. coli. C, intrinsic fluorescence exhibited by WT-RelM. tb and MT-RelM. tb. Emission spectra were recorded at λex of 280 nm. The inset shows a structure-based sequence comparison of the catalytic loop of RelS. eq, RelM. tb, and RelAE. coli, with the numbers corresponding to that of RelS. eq.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: A, interchanging RXKD and EXDD motifs in Rel proteins affects (p)ppGpp synthesis. (p)ppGpp synthesis was assayed for the indicated wild type and mutant proteins using either GTP or GDP along with [γ-32P]ATP. The activity of MT-RelAE. coli is plotted while considering WT-RelAE. coli activity to be 100%. The activities of WT-RelM. tb and MT-RelM. tb are also plotted similarly. B, intrinsic fluorescence exhibited by WT-RelAE. coli, MT-RelAE. coli, and W260A mutant of WT-RelAE. coli. C, intrinsic fluorescence exhibited by WT-RelM. tb and MT-RelM. tb. Emission spectra were recorded at λex of 280 nm. The inset shows a structure-based sequence comparison of the catalytic loop of RelS. eq, RelM. tb, and RelAE. coli, with the numbers corresponding to that of RelS. eq.
Mentions: All the values were determined in the absence of cofactors such as mRNA, tRNA, and/or ribosomes or denaturants such as methanol and detergents, unlike the earlier studies (5, 15). Values obtained from three independent experiments were used for calculating the S.D. MT-RelM. tb (EXDD) was not used here, as (p)ppGpp synthesis was reduced by 90% (see Fig. 3A).

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