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

• Published in: Journal of Bacteriology 2014, Vol.196 (1), p.90-99
• Main Authors: Kumar, Sanjay, Kanudia, Pavitra, Karthikeyan, Subramanian, Chakraborti, Pradip K
• Format: Article
• Language: English
• Subjects: active sites; Amidohydrolases - genetics; Amidohydrolases - metabolism; amino acid sequences; Bacteria; Bacteriology; cysteine; dissociation; DNA Mutational Analysis; enzyme activity; Enzyme Inhibitors - metabolism; hydrogen peroxide; Hydrogen Peroxide - metabolism; Inhibitory Concentration 50; ionization; metalloproteinases; methionine; mutants; Mutation; Mycobacterium tuberculosis; Mycobacterium tuberculosis - enzymology; Mycobacterium tuberculosis - genetics; Mycobacterium tuberculosis - metabolism; oxidants; Oxidation; Oxidative Stress; oxygen; Peptides; polypeptides; Protein synthesis; proteins; Salmonella; Salmonella enterica; Salmonella enterica subsp. enterica serovar Typhimurium; Salmonella Typhimurium; Salmonella typhimurium - enzymology; Salmonella typhimurium - genetics; Salmonella typhimurium - metabolism; sequence analysis
• ISSN: 0021-9193; 1098-5530; 1067-8832
• DOI: 10.1128/JB.00916-13

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.