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Identification of essential residues within Lit, a cell death peptidase of Escherichia coli K-12.

Copeland, Nikki and Bingham, Ryan and Georgiou, Theonie and Cooper, Peter and Kleanthous, Colin (2004) Identification of essential residues within Lit, a cell death peptidase of Escherichia coli K-12. Biochemistry, 43 (24). pp. 7948-7953. ISSN 0006-2960

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Abstract

Bacteriophage exclusion is a suicide response to viral infection. In strains of Escherichia coli K-12 infected with T4 phage this process is mediated by the host-encoded Lit peptidase. Lit is activated by a unique sequence in the major head protein of the T4 phage (the Gol sequence) which then cleaves site-specifically the host translation factor EF-Tu, ultimately leading to cell death. Lit has very low sequence identity with other peptidases, with only a putative metallopeptidase motif, H(160)EXXH, giving an indication of its catalytic activity. The aim of the present study was to ascertain if Lit is a metallopeptidase, identify residues essential for Lit activity, and probe the involvement of the Gol sequence in the activation of enzymatic activity. Lit activity was inhibited by the zinc chelator 1,10-phenanthroline, consistent with the suggestion that it is a metallopeptidase. Preliminary covalent modification experiments found that Lit was susceptible to inactivation by diethyl pyrocarbonate, with about three histidines reversibly modified, one of which was found to be essential for proteolytic activity. Subsequently, 13 mutants of the Lit enzyme were constructed that included all 10 histidines as well as other residues within the metallopeptidase motif. This demonstrated that the residues within the HEXXH motif are required for Lit activity and further defined the essential catalytic core as H(160)EXXHX(67)H, with additional residues such as His169 being important but not essential for activity. Kinetic analysis of Lit activation by a synthetic Gol peptide highlighted that elevated concentrations of the peptide (>10-fold above activation K(M)) are inhibitory to Lit, with this effect also seen in partially active Lit mutants. The susceptibility of Lit to inhibition by its own activating peptide suggests that the Gol sequence may be able to bind nonproductively to the enzyme at high concentration. We discuss these data in the context of the currently understood models for Gol-mediated activation of the Lit peptidase and its mechanism of action.

Item Type: Article
Journal or Publication Title: Biochemistry
Subjects:
Departments: Faculty of Health and Medicine > Biomedical & Life Sciences
ID Code: 52425
Deposited By: ep_importer_pure
Deposited On: 30 Jan 2012 09:16
Refereed?: Yes
Published?: Published
Last Modified: 09 Apr 2014 23:02
Identification Number:
URI: http://eprints.lancs.ac.uk/id/eprint/52425

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