PURIFICATION, CHARACTERIZATION, AND INHIBITION OF PEPTIDE DEFORMYLASEFROM ESCHERICHIA-COLI

Peptide deformylase (EC 3,5.1.31) catalyzes the removal of a formyl gr oup from the N-termini of nascent ribosome-synthesized polypeptides, a n obligatory step during protein maturation in eubacteria, Since its d iscovery in crude Escherichia coli extracts 3 decades ago, the deformy lase has resisted all attempts of purification or characterization due to its extraordinary lability. By placing the coding sequence (def ge ne) of Escherichia coli deformylase behind a bacteriophage T7 promoter ; we have, however, been able to overexpress this deformylase in Esche richia coli. Overproduction has allowed the purification of >50 mg of deformylase enzyme from each liter of cell culture. Purified deformyla se is highly active toward N-formylated peptide substrates. A new, sen sitive assay for the deformylase has been developed by measuring the a mount of released formate using a formate dehydrogenase; This has allo wed for the assessment of the catalytic properties of peptide deformyl ase using a series of synthetic N-formylated peptides as substrates. T he deformylase exhibits strong preference for an L-methionine or the i sosteric norleucine at the N-terminus of a substrate and has broad spe cificity for the rest of the residues, Small divalent metal chelators strongly inhibit the E. call deformylase. In particular: certain 1,2- and 1,3-dithiol compounds act as potent, lime-dependent inhibitors of the peptide deformylase.