Characterization of peptidyl-tRNA hydrolase encoded by open reading frame Rv1014c of Mycobacterium tuberculosis H37Rv.
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-[(3)H]-lysine from diacetyl-[(3)H]-lysyl-tRNA(Lys) with Michaelis-Menten kinetic parameters of K (m)=0.7+/-0.2 microM 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 degrees C, demonstrating the in vivo activity of MtPth. In addition, at 39 degrees C, over-expression of MtPth in AA7852 cells allowed the cells to remain viable in the presence of up to 200 microg/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.
Bal
Naresh C
NC
Molecular and Structural Biology, Central Drug Research Institute, Lucknow, India.
Agrawal
Himanshu
H
Meher
Akshaya K
AK
Arora
Ashish
A
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D017124:Conserved Sequence; D004795:Enzyme Stability; D004917:Erythromycin / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics*; D019906:Nuclear Magnetic Resonance, Biomolecular; D016366:Open Reading Frames / Q000235:genetics*; D017510:Protein Folding; D016415:Sequence Alignment; D040681:Structural Homology, Protein; D013696:Temperature
D000906:Antibodies; D004917:Erythromycin; D002265:Carboxylic Ester Hydrolases; C019674:aminoacyl-tRNA hydrolase