Characterization of peptidyl-tRNA hydrolase encoded by open reading frame Rv1014c of Mycobacterium tuberculosis H37Rv
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Naresh C. Bal
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 Km=0.7±0.2 μM and kcat=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.
©2007 by Walter de Gruyter Berlin New York
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Artikel in diesem Heft
- Mid-region parathyroid hormone-related protein (PTHrP) and gene expression of MDA-MB231 breast cancer cells
- Characterization of peptidyl-tRNA hydrolase encoded by open reading frame Rv1014c of Mycobacterium tuberculosis H37Rv
- Calcium signalling in the regulation of PGC-1α, PDK4 and HKII mRNA expression
- Expression of the protein phosphatase 1 inhibitor KEPI is downregulated in breast cancer cell lines and tissues and involved in the regulation of the tumor suppressor EGR1 via the MEK-ERK pathway
- Nitric oxide modulates expression of extracellular matrix genes linked to fibrosis in kidney mesangial cells
- Discovery and characterization of human antibody inhibitors of pregnancy-associated plasma protein-A
- The protease domain of procollagen C-proteinase (BMP1) lacks substrate selectivity, which is conferred by non-proteolytic domains
- Two α subunits and one β subunit of meprin zinc-endopeptidases are differentially expressed in the zebrafish Danio rerio
- Increase in kinins on post-exercise hypotension in normotensive and hypertensive volunteers
- Inhibition of cathepsin L-like proteases by cathepsin V propeptide
- Clinical chemistry reference database for Wistar rats and C57/BL6 mice
