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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 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.

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