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

• Published in: Biochemistry (Easton) 2005-07, Vol.44 (29), p.9913-9923
• Main Authors: Avarbock, Andrew, Avarbock, David, Teh, Jiah-Shin, Buckstein, Michael, Wang, Zhi-mei, Rubin, Harvey
• Format: Article
• Language: English
• Subjects: Binding Sites; Catalytic Domain; Enzyme Stability; Hydrolysis; Ligases - biosynthesis; Ligases - chemistry; Ligases - genetics; Ligases - metabolism; Magnesium - chemistry; Multienzyme Complexes - biosynthesis; Multienzyme Complexes - chemistry; Multienzyme Complexes - genetics; Multienzyme Complexes - metabolism; Mycobacterium tuberculosis - enzymology; Mycobacterium tuberculosis - genetics; Peptide Fragments - chemistry; Peptide Fragments - genetics; Pyrophosphatases - biosynthesis; Pyrophosphatases - chemistry; Pyrophosphatases - genetics; Pyrophosphatases - metabolism; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0505316

The dual-function RelMtb 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 RelMtb 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 RelMtb 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 RelMtb fragment proteins have basal activities similar to that of full-length RelMtb, but are no longer regulated by the previously described RelMtb activating complex (RAC). Residues within the C-terminus of RelMtb (D632A and C633A) are shown to have a role in interaction with the RAC. Additionally, size exclusion chromatography indicates RelMtb 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.