title: Identification of crucial amino acids of bacterial Peptide deformylases affecting enzymatic activity in response to oxidative stress.
abstract: 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.
authors.affiliation: CSIR-Institute of Microbial Technology, Chandigarh, India.
authors.forename: Sanjay
authors.initials: S
authors.lastname: Kumar
authors.forename: Pavitra
authors.initials: P
authors.lastname: Kanudia
authors.forename: Subramanian
authors.initials: S
authors.lastname: Karthikeyan
authors.forename: Pradip K
authors.initials: PK
authors.lastname: Chakraborti
mesh_terms: D000581:Amidohydrolases / Q000235:genetics* / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004791:Enzyme Inhibitors / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D020128:Inhibitory Concentration 50; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D018384:Oxidative Stress; D012486:Salmonella typhimurium / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D006861:Hydrogen Peroxide; D000581:Amidohydrolases; C084507:peptide deformylase