Three consecutive arginines are important for the mycobacterial peptide deformylase enzyme activity

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.

FIGURE 1.

Deletion analysis of insertion region of mPDF. A, alignment of amino acid residues between motifs I and II from PDFs of different mycobacterial species (residues 46–111), E. coli (residues 41–96), and Staphylococcus aureus (residues 55–116) showing three consecutive arginines (shaded). The asterisks and dots denote identical and similar amino acids, respectively. M. tb, M. tuberculosis; M. smeg, M. smegmatis. B, deletion scheme. WT, wild-type mPDF; ΔIR, deletion of MTARRRGVVINP residues; ΔMTA, deletion of MTA residues; ΔID, deletion of MTARRR residues. C, assessment of deformylase activity of deletion mutants. Deformylase assays were carried out with the indicated amounts of wild-type or mutant proteins using 5 mm N-formyl-Met-Ala as the substrate. Inset, Western blot of wild-type and mutant proteins (1 μg/slot) using rabbit polyclonal anti-serum against recombinant mPDF.

FIGURE 2.

Three consecutive arginines of the mPDF insertion region are crucial for deformylase activity. A, kinetic analyses of deformylase activities of mutant proteins. Inset, enzyme activities as a function of amount of protein using 5 mm substrate. B, comparison of enzymatic stabilities of wild-type (WT) and mutant proteins at 30 °C. C, effect of H₂O₂ on the deformylase activity of mutants. Wild-type or mutant proteins were preincubated with or without H₂O₂ (final concentration, 500 mm) at 30 °C for 15 min. This was followed by an enzyme assay using 5 mm substrate.

FIGURE 3.

Effect of mutations on the secondary structure of mPDF. Far-UV CD spectra of wild-type (WT) and mutant proteins (R77K, R78K, R79K, and R77K/R78K/R79K) in 20 mm phosphate buffer, pH 7.4, were obtained between wavelengths 250 and 187 nm. The Ni²⁺-nitrilotriacetic acid-purified protein samples (∼0.2 mg/ml) were dialyzed to remove immidazole and were used for spectroscopic analysis.

FIGURE 4.

Model of mPDF superimposed on ePDF. The superimposition shows that the core regions are conserved in mPDF (blue) and ePDF (pink) along with metal ion (red) and Met-Ala-Ser tripeptide (green). Arginines (R77K/R18K/R79K) are away from the substrate binding pocket. The inset shows the position of residues (yellow) interacting with tripeptide and metal ion in PDF.

FIGURE 5.

Comparison of r.m.s. deviations (RMSD) as a function of time for wild-type (black) and mutant (gray) mPDF.

FIGURE 6.

Relative probability plots for the distances observed during the course of dynamics between the atoms of wild type (solid line) and mutant (dashed line) mPDF. A, C_δ1 and C_ε; B, C_δ2 and C_ε of Leu¹⁰⁷ and Met¹⁴⁵, respectively; C, C_δ1 and NH₁; D, C_δ2 and NH₁; E, C_δ1 and NH₂; F, C_δ2 and NH₂ of Leu¹⁰⁷ and Arg¹⁴⁴, respectively.

FIGURE 7.

Stereoview of the substrate binding pocket residues of mPDF. The conformation of the substrate binding pocket residues of the averaged structure from simulations for the wild type and the mutant are shown in a stick model and labeled. Dashed lines (cyan) indicate the distance between the atoms in Å units. The figure was generated using PYMOL (29).

FIGURE 8.

PTO-modified antisense oligodeoxynucleotides against the insertion region of mPDF affect mycobacterial growth. M. smegmatis or E. coli K12 cultures were grown in the absence or presence of PS-ODNs (10 μm). A, effect of PS-ODN-(74–83) on the growth of M. smegmatis. A, inset, representative experiment showing the effect of PS-ODNAM-(76–85) (all bases PTO-modified) on M. smegmatis growth. B, effect of PS-ODN-(74–83) on the growth of E. coli. C, permeability of flourescein-conjugated PS-ODN-(74–83) within M. smegmatis cells. Culture was grown in the absence (top) or presence (bottom) of PS-ODNF-(74–83) for 24 h and visualized under a confocal microscope. D, expression of M. smegmatis native PDF in response to PS-ODN-(74–83) treatment. Soluble fractions of cell lysates were prepared from treated or untreated cells. Following the protein estimation, samples were resolved by 12% SDS-PAGE, transferred to nitrocellulose membrane, and subjected to Western blotting using anti-mPDF polyclonal antibody. A representative experiment shows Ponceau S staining of nitrocellulose membrane after transfer (top) and following development of Western blot using the ECL detection system (bottom). The reproducibility of the results was checked from three independent experiments. The arrowhead indicates the position of the specific PDF band. The numbers denote the molecular weight (in kDa) of the marker.