Oxygen-mediated Inactivation of Peptide Deformylase

Abstract

Peptide deformylase catalyzes the removal of the Nformyl group from newly synthesized polypeptides in prokaryotes. Its essential character and unique presence in prokaryotes make it an attractive target for antibacterial chemotherapy. However, purification and characterization of the peptide deformylase have remained a major challenge because this enzyme is extraordinarily labile under a variety of conditions (t 1/2 ϳ1 min at room temperature Protein synthesis in prokaryotes initiates with an N-formylmethionyl-tRNA i , resulting in N-terminal formylation of all nascent polypeptides (1). Peptide deformylase catalyzes the subsequent removal of the formyl group from the majority of bacterial proteins (2)(3)(4). Although the precise functions of the formylation and deformylation steps remain somewhat obscure, genetic studies have shown that the peptide deformylase activity is essential for bacterial survival (5, 6). Because peptide deformylase is apparently absent in eukaryotic systems (7,8), it provides a potential target for a novel antibacterial strategy (5). Very recently, biochemical studies from this laboratory have established that peptide deformylase represents an intriguing new class of amide hydrolyase, which utilizes a ferrous ion as the catalytic metal (7). The unusual structural feature of peptide deformylase and its potential application in antibiotic studies warrant a detailed mechanistic investigation of this class of enzymes.Although the deformylase activity was recognized three decades ago (2-4), the extraordinary lability of this enzyme has long prevented its purification and characterization. It was reported that even mild procedures such as dialysis, Sephadex G-100 or DEAE-cellulose chromatography, or ultracentrifugation on sucrose gradient resulted in almost quantitative loss of enzymatic activity (2, 3). At 37°C, the deformylase in a crude cell lysate lost activity with a half-life of 60 s (3). We have recently overexpressed the deformylase from Escherichia coli and purified the active enzyme to homogeneity (7, 9). Unfortunately, the purified enzyme remains highly unstable, complicating the quantitative evaluation of its activity. In this work, we have found that the instability of peptide deformylase is because of oxidation of the catalytic Fe 2ϩ ion into the catalytically inactive ferric ion by atmospheric O 2 . A simple experimental procedure has been developed to preserve the deformylase activity. These findings will greatly facilitate future mechanistic investigations of the peptide deformylase as well as high throughput screening for deformylase inhibitors. EXPERIMENTAL PROCEDURESMaterials-Glucose oxidase (EC 1.1.3.4), superoxide dismutase, and catalase (EC 1.11.1.6) from Aspergillus niger were purchased from Fluka (Ronkonkoma, NY). Aeromonas aminopeptidase and all chemicals were from Sigma Chemical Company.Buffers-All purification buffers were exhaustively degassed under vacuum and then sparged with argon prior to use. Buffer A, 20 mM potassium phosphate, pH 8.0, 10 mM NaCl, 1% Triton X...

An Unusual Peptide Deformylase Features in the Human Mitochondrial N-terminal Methionine Excision Pathway

“…1). Our results have shown that the Cys 43 and Glu 91 substitutions are responsible for the unusual behavior of HsPDF, as the replacement of these two residues by Gly and Leu, respectively, makes HsPDF as active as plant PDF1As (Table II). The side chains of residues 43 and 91 are located in close proximity (within 4.5 Å) in the crystal structure of known PDF1s (24).…”

“…However, we cannot exclude the possibility that this domain is also involved in binding to mitoribosomes, like the additional N-terminal domain of MAP1As to cytosolic ribosomes. All animal PDFs display systematic substitutions altering crucial amino acids in motifs 1 and 2: Gly 43 , Ala 47 , and Leu 91 (Fig. 1).…”

“…11 and 23). The purified ⌬N39 form contained zinc bound in a stoichiometric manner, suggesting that zinc had replaced the physiological metal cation, which was probably iron, as in PDF1Bs (11,20,43).…”

A Role for Iron in an Ancient Carbonic Anhydrase

“…Furthermore, E. coli cultured in unsupplemented medium produced Cam with 7-fold more iron than zinc, although metals analysis of the medium showed it contained 1.6 ppm of iron and 3. (21). Use of strictly anaerobic conditions to prevent oxidation also lead to the identification of Fe 2ϩ as the active site metal in the previously reported Zn 2ϩ -containing methionyl aminopeptidase from E. coli (22) (23).…”

The Crystal Structure of Mitochondrial (Type 1A) Peptide Deformylase Provides Clear Guidelines for the Design of Inhibitors Specific for the Bacterial Forms

“…11). The ferrous cation of PDF is extremely sensitive to oxidation, making the enzyme unstable (9,12). This cation can be replaced by Ni 2ϩ (9,13) or Co 2ϩ (14) to give stable variants with little or no loss of catalytic activity.…”