Oxidation of active site cysteine leads to inactivation of peptide deformylase from Salmonella enterica

Abstract

Peptide deformylase (PDF) is an essential bacterial enzyme involved in first step of N-methionine excision (NME) pathway during bacterial protein synthesis. In first step of NME, N-formyl group of nascent polypeptide chains is removed by PDF. PDF is a metallo-protease where metal cofactor is co-ordinated to a Cys and two His residues. We cloned and expressed this iron containing metallo-protease from Salmonella typhimurium (sPDF). We characterized the sPDF which is a mononuclear iron containing enzyme that displayed optimal in vitro activity in the presence of oxidation preventing agent like catalase. To have an insight into the role of metal ion in catalase dependent enzyme activity, we generated surrogate sPDF-Ni²⁺ and sPDF-Co²⁺. Interestingly, these proteins also showed catalase requirement for optimum enzyme activity. Thus our results argue the presence of the target (amino acid) of oxidation in the protein itself that might be crucial for the activity of the enzyme. To ascertain this aspect, we examined the oxidation status of active site cysteine of sPDF by mass spectrometry. Our results indicated that the direct oxidation/over-oxidation of active site cysteine is responsible for inactivation of sPDF protein. Furthermore, a comparison of PDF sequences from Gram-negative bacteria revealed the presence of this cysteine throughout the lineage. Thus, our results per se are indicative of a similar behaviour of all these Gram-negative peptide deformylase proteins.