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Abstract:
Colicin M (Cma), a protein toxin from E. coli, is a novel phosphatase concerning sequence, structure and substrate specificity. It is is imported into the periplasm of sensitive cells via a receptor-dependent energy- coupled process. E. coli and closely related strains are killey by inhibition of murein biosynthesis; Cma cleaves the phosphate ester bond between the lipid carrier and the murein precursor. This mode of action is unique for Cma. With 271 amino acid residues, it is the smallest of all known colicins. Its fold is unique among colicins and even among all known proteins. The protein forms a compact structure, which makes it difficult to delineate the functional domains which are well-separated in most other colicins [1]. To study these functional domains of Cma, mutants in the various predicted domains were isolated and characterized with special emphasis on the activity domain. The active site is located in a surface-exposed region. Conversion of Asp226 to Glu, Asn, or Ala inactivated Cma. This residue is exposed at the Cma surface and is surrounded by Asp225, Tyr228, Asp229, His235 and Arg236; replacement of each residue with alanine inactivated Cma. We propose that Asp226 directly participates in phosphate ester hydrolysis and that the surrounding residues contribute to the active site. All these residues are strongly conserved in Cma-like proteins of other species. Moreover, we found that the hydrophobic helix 1, that extends from the compact Cma structure, binds the toxin to the FhuA receptor in the outer membrane and is thereby involved in its uptake [3]. Killing of cells by Cma strictly depends on the periplasmic peptidyl prolyl cis/trans isomerase/chaperone FkpA [4]. Because of its compact structure the colicin must unfold during translocation across the outer membrane und refold in the periplasm to be toxic. This is supported by FkpA that presumably assists in refolding by cis/trans isomerisation of one or a few prolyl bonds. To identify the Cma prolyl bonds targeted by FkpA, we replaced the 15 proline residues individually with alanine and found four mutants with reduced activities. P107A displayes 10%, P129A, P176A and P260A show 1% activity. Three of them were not imported, the remaining P176A mutant is structural identical to wild-type Cma which makes it unlikely that the mutation changes the phosphatase active site that is located far from this proline residue. In an in vitro peptide assay FkpA isomerized the Cma prolyl bond Phe175-Pro176 at a high rate. These results suppose that this bond is most likely targeted by FkpA in the activation of Cma in the periplasm [4].
