Functional Regulation of the Opposing (p)ppGpp Synthetase/Hydrolase Activities of Rel_Mtb from Mycobacterium tuberculosis

Abstract

The dual-function Rel(Mtb) protein from Mycobacterium tuberculosis catalyzes both the synthesis and hydrolysis of (p)ppGpp, the effector of the stringent response. In our previous work [Avarbock, D., Avarbock, A., and Rubin, H. (2000) Biochemistry 39, 11640], we presented evidence that the Rel(Mtb) protein might catalyze its two opposing reactions at distinct active sites. In the study presented here, we purified and characterized fragments of the 738-amino acid Rel(Mtb) protein and confirmed the hypothesis that amino acid fragment 1-394 contains both synthesis and hydrolysis activities, amino acid fragment 87-394 contains only (p)ppGpp synthesis activity, and amino acid fragment 1-181 contains only (p)ppGpp hydrolysis activity. Mutation of specific residues within fragment 1-394 results in the loss of synthetic activity and retention of hydrolysis (G241E and H344Y) or loss of hydrolytic activity with retention of synthesis (H80A and D81A). The C-terminally cleaved Rel(Mtb) fragment proteins have basal activities similar to that of full-length Rel(Mtb), but are no longer regulated by the previously described Rel(Mtb) activating complex (RAC). Residues within the C-terminus of Rel(Mtb) (D632A and C633A) are shown to have a role in interaction with the RAC. Additionally, size exclusion chromatography indicates Rel(Mtb) forms trimers and removal of the C-terminus results in monomers. The C-terminal deletion, 1-394, which exists as a mixture of monomers and trimers, will dissociate from the trimer state upon the addition of substrate. Furthermore, the trimer state of fragment 1-394 appears to be a catalytically less efficient state than the monomer state.

The Significance of EXDD and RXKD Motif Conservation in Rel Proteins

“…On the other hand, RXKD-containing WTRel M. tb and MT-RelA E. coli show a preference for GTP over GDP, shown by their lower K m and higher V max for GTP. These values are not in agreement with the kinetic constants reported earlier (5,15) for the wild type proteins RelA E. coli and Rel M. tb , as they were determined in the presence of cofactors (mRNA, tRNA, and/or ribosome) or denaturants (methanol or detergents) that were absent in our study. Nevertheless, these results demonstrate the importance of the motifs RXKD/EXDD in governing substrate specificities in Rel proteins.…”

“…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.…”

Polyphosphate and stress response in mycobacteria

“…Mycobacteria have only one enzyme responsible for both (p)ppGpp biosynthesis and hydrolysis (Avarbock et al, 2005). The best characterized of these enzymes is Rel Mtb in M. tuberculosis.…”

A Truncated Form of SpoT, Including the ACT Domain, Inhibits the Production of Cyclic Lipopeptide Arthrofactin, and Is Associated with Moderate Elevation of Guanosine 3′,5′-Bispyrophosphate Level inPseudomonassp. MIS38

“…26) Deleting the C-terminal region of Rel mtb from Mycobacterium tuberculosis makes it non-responsive to the ribosome, but in this case the enzyme activity does not change. 27) Our present study indicates that C-terminal truncation of MIS38 SpoT increases the level of arthrofactin production (spoT delC) ( Table 2). It is unclear how the enzyme activity of MIS38 SpoT is changed by C-terminal deletion, since the ppGpp levels of the wild type MIS38 and the spoT-delC strain are below the detectable range (data not shown).…”

“…In a study of M. tuberculosis, a role in oligomerization has been suggested for the C-terminal region of Rel mtb . 27) If SpoT Cter can be folded into a stable domain structure in MIS38 and can modulate the enzyme assembly of SpoT, the balance of the synthesis and hydrolysis of (p)ppGpp is influenced.…”