Functional Regulation of the Opposing (p)ppGpp Synthetase/Hydrolase Activities of RelMtb from Mycobacterium tuberculosis
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- 作者：Andrew Avarbock ; David Avarbock ; Jiah-Shin Teh ; Michael Buckstein ; Zhi-mei Wang ; and Harvey Rubin
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：July 26, 2005
- 年：2005
- 卷：44
- 期：29
- 页码：9913 - 9923
- 全文大小：428K
- 年卷期：v.44,no.29(July 26, 2005)
- ISSN：1520-4995
文摘
The dual-function RelMtb protein from Mycobacterium tuberculosis catalyzes both the synthesisand 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 RelMtbprotein might catalyze its two opposing reactions at distinct active sites. In the study presented here, wepurified and characterized fragments of the 738-amino acid RelMtb protein and confirmed the hypothesisthat amino acid fragment 1-394 contains both synthesis and hydrolysis activities, amino acid fragment87-394 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 ofsynthetic activity and retention of hydrolysis (G241E and H344Y) or loss of hydrolytic activity withretention of synthesis (H80A and D81A). The C-terminally cleaved RelMtb fragment proteins have basalactivities similar to that of full-length RelMtb, but are no longer regulated by the previously describedRelMtb activating complex (RAC). Residues within the C-terminus of RelMtb (D632A and C633A) areshown to have a role in interaction with the RAC. Additionally, size exclusion chromatography indicatesRelMtb 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 additionof substrate. Furthermore, the trimer state of fragment 1-394 appears to be a catalytically less efficientstate than the monomer state.