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J. Biol. Chem., Vol. 283, Issue 35, 23754-23764, August 29, 2008

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Three Consecutive Arginines Are Important for the Mycobacterial Peptide Deformylase Enzyme Activity^*

From the Institute of Microbial Technology, Council of Scientific and Industrial Research, Sector 39A, Chandigarh 160 036, India

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.

Received for publication, November 27, 2007 , and in revised form, June 20, 2008.

^* This work was supported by Council of Scientific and Industrial Research, New Delhi (under network program SMM003) and Department of Biotechnology (New Delhi, India) and financial support in the form of research fellowships (to R. S., P. K., H. H. D., and M. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1 and Figs. 1 and 2.

¹ Present address: Dept. of Biochemistry and Cell Biology, Georgetown University Medical Centre, Washington, D. C. 20007.

² To whom correspondence should be addressed. Tel.: 91-172-2690751; Fax: 91-172-2690585; E-mail: pradip{at}imtech.res.in.

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