Characterization of peptidyl-tRNA hydrolase encoded by open reading frame Rv1014c of Mycobacterium tuberculosis H37Rv : Biological Chemistry Jump to ContentJump to Main Navigation
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Biological Chemistry

Editor-in-Chief: Brüne, Bernhard

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Characterization of peptidyl-tRNA hydrolase encoded by open reading frame Rv1014c of Mycobacterium tuberculosis H37Rv

Naresh C. Bal1 / Himanshu Agrawal2 / Akshaya K. Meher3 / Ashish Arora4

1Molecular and Structural Biology, Central Drug Research Institute, Lucknow 226 001, India
The first two authors contributed equally to this work.

2Molecular and Structural Biology, Central Drug Research Institute, Lucknow 226 001, India

3Molecular and Structural Biology, Central Drug Research Institute, Lucknow 226 001, India

4Molecular and Structural Biology, Central Drug Research Institute, Lucknow 226 001, India

Corresponding author

Citation Information: Biological Chemistry. Volume 388, Issue 5, Pages 467–479, ISSN (Online) 14316730, ISSN (Print) 14374315, DOI: 10.1515/BC.2007.057, May 2007

Publication History

Received:
2006-11-02
Accepted:
2007-02-13
Published Online:
2007-05-01

Abstract

The enzyme peptidyl-tRNA hydrolase (Pth, EC 3.1.1.29) is essential for the viability of bacteria. The ORF Rv1014c of Mycobacterium tuberculosis H37Rv, designated as the mtpth gene, was cloned and over-expressed and the product was purified. Generation of polyclonal antibodies against the purified recombinant protein, termed MtPth, facilitated detection of endogenously expressed MtPth in M. tuberculosis H37Rv cell lysate. MtPth could release diacetyl-[3H]-lysine from diacetyl-[3H]-lysyl-tRNALys with Michaelis-Menten kinetic parameters of K m=0.7±0.2 μM and k cat=1.22±0.2 s-1. Transformation of a pTrc99c/mtpth vector allowed the growth of E. coli thermosensitive Pth(ts) mutant strain AA7852 at the non-permissive temperature of 42°C, demonstrating the in vivo activity of MtPth. In addition, at 39°C, over-expression of MtPth in AA7852 cells allowed the cells to remain viable in the presence of up to 200 μg/ml erythromycin. A 3D fold based on NMR and a structural model based on the E. coli Pth crystal structure were generated for MtPth. The essential nature of conserved active-site residues N12, H22 and D95 of MtPth for catalysis was demonstrated by mutagenesis and complementation in E. coli mutant strain AA7852. Thermal and urea/guanidinium chloride (GdmCl)-induced unfolding curves for MtPth indicate a simple two-state unfolding process without any intermediates.

Keywords: complementation; enzyme activity; molecular modeling; mutagenesis; peptidyl-tRNA hydrolase; thermal unfolding

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