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Peptide deformylase (PDF) is an essential bacterial metalloprotease involved in deformylation of N-formyl group from nascent polypeptide chains during protein synthesis. Iron-containing variants of this enzyme from Salmonella enterica serovar Typhimurium (sPDF) and Mycobacterium tuberculosis (mPDF), although inhibited by oxidizing agents like H2O2, exhibited strikingly different 50% inhibitory concentrations (IC50s) that ranged from nanomolar (sPDF) to millimolar (mPDF) levels. Furthermore, the metal dissociation rate was higher in sPDF than mPDF. We hypothesized that a restriction in entry of environmental oxygen or oxidizing agents into the active site of mPDF might be the cause for such discrepancies between two enzymes. Since the active-site residues of the two proteins are similar, we evaluated the role of the oxidation-prone noncatalytic residue(s) in the process. Amino acid sequence analysis revealed that Cys-130 of sPDF corresponds to Met-145 of mPDF and that they are away from the active sites. Swapping methionine with cysteine in mPDF, the M145C protein displayed a drastic decrease in the IC50 for H2O2 and an increased metal dissociation rate compared to the wild type. Matrix-assisted laser desorption ionization (MALDI) analysis of a trypsin-digested fragment containing Cys-145 of the M145C protein also indicated its increased susceptibility to oxidation. To incorporate residues identical to those of mPDF, we created a double mutant of sPDF (C130M-V63C) that showed increased IC50 for H2O2 compared to the wild type. Interestingly, the oxidation state of cysteines in C130M-V63C was unaffected during H2O2 treatment. Taken together, our results unambiguously established the critical role of noncatalytic cysteine/methionine for enzymatic sensitivity to H2O2 and, thus, for conferring behavioral distinction of bacterial PDFs under oxidative stress conditions.

Identification of Crucial Amino Acids of Bacterial Peptide Deformylases Affecting Enzymatic Activity in Response to Oxidative Stress