Kumar, Sanjay and Kanudia, Pavitra and Karthikeyan, Subramanian and Chakraborti, Pradip K (2014) Identification of crucial amino acids of bacterial Peptide deformylases affecting enzymatic activity in response to oxidative stress. Journal of bacteriology, 196 (1). pp. 90-99. ISSN 1098-5530
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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.
| Item Type: | Article |
|---|---|
| Additional Information: | Copyright of this article belongs to American Society for Microbiology. |
| Uncontrolled Keywords: | amino acids;oxidative stress |
| Subjects: | Q Science > QR Microbiology |
| Depositing User: | Dr. K.P.S.Sengar |
| Date Deposited: | 30 May 2014 03:59 |
| Last Modified: | 30 May 2014 03:59 |
| URI: | http://crdd.osdd.net/open/id/eprint/1506 |
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