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We report the overproduction of the immunity protein for the DNase colicin E9 and its characterization both in vivo and in vitro. The genes for colicin immunity proteins are normally co-expressed from Col plasmids with their corresponding colicins. In the context of the enzymatic colicins, the two proteins form a complex, thereby protecting the host bacterium from the antibiotic activity of the colicin. This complex is then released into the medium, whereupon the colicin alone translocates (through the appropriate receptor) into sensitive bacterial strains, resulting in bacterial cell death. The immunity protein for colicin E9 (Im9) has been overproduced in a bacterial host in the absence of its colicin, to enable sufficient material to be isolated for structural studies. As a prelude to such studies, the in-vivo and in-vitro properties of overproduced Im9 were analysed. Electrospray mass spectrometry verified the molecular mass of the purified protein and analytical ultracentrifugation indicated that the native protein approximates a symmetric monomer. Fluorescence-enhancement and gel-filtration experiments show that purified Im9 binds to colicin E9 in a 1:1 molar ratio and that this binding neutralizes the DNase activity of the colicin. These results lay the foundations for a full biophysical and structural characterization of the colicin E9 DNase inhibitor protein, Im9.

Bacterial resistance to the antibiotic chloramphenicol is normally mediated by chloramphenicol acetyltransferase (CAT), which utilizes acetyl coenzyme A as the acyl donor in the inactivation reaction. 3-(Bromoacetyl)chloramphenicol, an analogue of the acetylated product of the forward reaction catalyzed by CAT, was synthesized as a probe for accessible and reactive nucleophilic groups within the active site. Extremely potent covalent inhibition was observed. Affinity labeling was demonstrated by the protection afforded by chloramphenicol at concentrations approaching Km for the substrate. Inactivation was stoichiometric, 1 mol of the inhibitor covalently bound per mole of enzyme monomer, with complete loss of both the acetylation and hydrolytic activities associated with CAT. N3-(Carboxymethyl)histidine was identified as the only alkylated amino acid, implicating the presence of a unique tautomeric form of a reactive imidazole group at the catalytic center. The proteolytic digestion of CAT modified with 3-(bromo[14C]-acetyl)chloramphenicol yielded three labeled peptide fractions separable by reverse-phase high-pressure liquid chromatography. Each peptide fraction was sequenced by fast atom bombardment mass spectrometry; the labeled peptide in each case was found to span the highly conserved region in the primary structure of CAT, which had been tentatively assigned as the active site. The rapid, stoichiometric, and specific alkylation of His-189, taken together with the high degree of conservation of the adjacent amino acid residues, strongly suggests a central role for His-189 in the catalytic mechanism of CAT.

The role of conserved Asp-199 in chloramphenicol acetyltransferase (CAT) has been investigated by site-directed mutagenesis. Substitution of Asp-199 by alanine results in a thermolabile mutant enzyme (Ala-199 CAT) with reduced kcat(13-fold) but similar Km values to wild type CAT. Replacement by asparagine gives rise to a thermostable mutant enzyme (Asn-199 CAT) with much reduced kcat(1500-fold). Furthermore, Asn-199 CAT shows anomalous inactivation kinetics with the affinity reagent 3-(bromo-acetyl)chloramphenicol. These results favor a structural role for Asp-199 rather than a catalytic one, in keeping with crystallographic evidence for involvement of Asp-199 in a tight salt bridge with Arg-18. Replacement of Arg-18 by valine results in a mutant enzyme (Val-18 CAT) with similar properties to Ala-199 CAT. The catalytic imidazole of His-19 appears to be conformationally constrained by hydrogen bonding between N1-H and the carbonyl oxygen of the same residue and by ring stacking with Tyr-25.