Purification, characterization, and inhibition of peptide deformylase from Escherichia coli.
Peptide deformylase (EC 3.5.1.31) catalyzes the removal of a formyl group from the N-termini of nascent ribosome-synthesized polypeptides, an obligatory step during protein maturation in eubacteria. Since its discovery in crude Escherichia coli extracts 3 decades ago, the deformylase has resisted all attempts of purification or characterization due to its extraordinary lability. By placing the coding sequence (def gene) of Escherichia coli deformylase behind a bacteriophage T7 promoter, we have, however, been able to overexpress this deformylase in Escherichia coli. Overproduction has allowed the purification of > 50 mg of deformylase enzyme from each liter of cell culture. Purified deformylase is highly active toward N-formylated peptide substrates. A new, sensitive assay for the deformylase has been developed by measuring the amount of released formate using a formate dehydrogenase. This has allowed for the assessment of the catalytic properties of peptide deformylase using a series of synthetic N-formylated peptides as substrates. The deformylase exhibits strong preference for an L-methionine or the isosteric norleucine at the N-terminus of a substrate and has broad specificity for the rest of the residues. Small divalent metal chelators strongly inhibit the E. coli deformylase. In particular, certain 1,2- and 1,3-dithiol compounds act as potent, time-dependent inhibitors of the peptide deformylase.
Rajagopalan
P T
PT
Department of Chemistry, Ohio State University, Columbus 43210, USA.
Datta
A
A
Pei
D
D
D000581:Amidohydrolases; D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D002413:Cations, Divalent; D002614:Chelating Agents / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008667:Metalloproteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000494:pharmacology
D002413:Cations, Divalent; D002614:Chelating Agents; D008667:Metalloproteins; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D000626:Aminopeptidases; D000581:Amidohydrolases; C084507:peptide deformylase