Three consecutive arginines are important for the mycobacterial peptide deformylase enzyme activity.
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.
Saxena
Rahul
R
Institute of Microbial Technology, Council of Scientific and Industrial Research, Chandigarh, India.
Kanudia
Pavitra
P
Datt
Manish
M
Dar
Haider Hussain
HH
Karthikeyan
Subramanian
S
Singh
Balvinder
B
Chakraborti
Pradip K
PK
D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D019943:Amino Acid Substitution; D001120:Arginine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002942:Circular Dichroism; D003198:Computer Simulation; D008958:Models, Molecular; D020125:Mutation, Missense; D020102:Mycobacterium smegmatis / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D017386:Sequence Homology, Amino Acid
D016376:Oligonucleotides, Antisense; D001120:Arginine; D000581:Amidohydrolases; C084507:peptide deformylase