Functional regulation of the opposing (p)ppGpp synthetase/hydrolase activities of RelMtb from Mycobacterium tuberculosis.
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.
Avarbock
Andrew
A
Division of Infectious Diseases, Department of Medicine, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104, USA.
Avarbock
David
D
Teh
Jiah-Shin
JS
Buckstein
Michael
M
Wang
Zhi-mei
ZM
Rubin
Harvey
H
D001665:Binding Sites; D020134:Catalytic Domain; D004795:Enzyme Stability; D006868:Hydrolysis; D008025:Ligases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry; D009097:Multienzyme Complexes / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D011755:Pyrophosphatases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism
D009097:Multienzyme Complexes; D010446:Peptide Fragments; D011994:Recombinant Proteins; C027627:guanosine-3',5'-bis(diphosphate) 3'-pyrophosphatase; D011755:Pyrophosphatases; D008025:Ligases; C028812:guanosine 3',5'-polyphosphate synthetases; D008274:Magnesium