Abstract
Genes encoding the peptide deformylase enzyme (def) are present in all eubacteria and are involved in the deformylation of the N-formyl group of newly synthesized polypeptides during protein synthesis. We compared the amino acid sequences of this enzyme in different mycobacterial species and found that they are highly conserved (76% homology with 62% identity); however, when this comparison was extended to other eubacterial homologs, it emerged that the mycobacterial proteins have an insertion region containing three consecutive arginine residues (residues 77-79 in Mycobacterium tuberculosis peptide deformylase (mPDF)). Here, we demonstrate that these three arginines are important for the activity of mPDF. Circular dichroism studies of wild-type mPDF and of mPDF containing individual conservative substitutions (R77K, R78K, or R79K) or combined substitutions incorporated into a triple mutant (R77K/R78K/R79K) indicate that such mutations cause mPDF to undergo structural alterations. Molecular modeling of mPDF suggests that the three arginines are distal to the active site. Molecular dynamics simulations of wild-type and mutant mPDF structures indicate that the arginines may be involved in the stabilization of substrate binding pocket residues for their proper interaction with peptide(s). Treatment with 5'-phosphothiorate-modified antisense oligodeoxyribonucleotides directed against different regions of def from M. tuberculosis inhibits growth of Mycobacterium smegmatis in culture. Taken together, these results hold out the possibility of future design of novel mycobacteria-specific PDF inhibitors.
Highlights
Peptidases acting at NH2 termini are unable to utilize formylated NH2 termini as substrates, the removal of the formyl group is mandatory for polypeptide maturation in all cases in which the removal of the NH2-terminal methionine is essential for protein folding or function
We found that M. tuberculosis peptide deformylase is an iron-containing protein that is enzymatically active as a multimer [15, 16]
We showed that the first six amino acids of the insertion region mentioned above play a role in stabilizing M. tuberculosis peptide deformylase (mPDF) and in imbuing it with resistance to oxidizing agents like H2O2 [16]
Summary
Materials—Restriction/modifying enzymes were obtained from New England Biolabs. ECL Western blotting detection kit, PCR DNA and gel band purification kit, and protein molecular weight markers were from GE Healthcare; the Expand long template PCR system was from Roche Applied Science; Herculase fusion DNA polymerase was from Stratagene; VentR DNA polymerase was from New England Biolabs; and plasmid DNA preparation kits were from Qiagen. Expression and Purification of Recombinant Proteins—The wild-type construct (pET-mPDF) and different mutant constructs were transformed into E. coli strain BL21(DE3) for overexpression and subsequent purification of NH2-terminally histidine-tagged fusion proteins. Overnight cultures of these clones (ϳ15 h at 37 °C in LB broth containing 50 g/ml kanamycin) were reinoculated and grown to an A600 of 0.8. Molecular dynamics simulations were performed on wild-type and mutant (R77K/R78K/R79K) structures Both proteins were immersed in a truncated octahedron box filled with about 8000 TIP3P water molecules. Analysis of production run (last 9 ns) of the simulations was done using the ptraj module of AMBER 9
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
