Identification of a Novel Inhibition Site in Translocase MraY Based upon the Site of Interaction with Lysis Protein E from Bacteriophage ϕX174. Issue 9 (4th June 2014)

• Record Type: Journal Article
• Authors: Rodolis, Maria T.; Mihalyi, Agnes; O'Reilly, Amy; Slikas, Justinas; Roper, David I.; Hancock, Robert E. W.; Bugg, Timothy D. H.
• Is Part Of: Chembiochem. Volume 15:Issue 9(2014)
• Page Start: 1300
• Page End: 1308
• Publication Date: 2014-06-04
• Subjects: Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pharmaceutical chemistry -- Periodicals; 572
• DOI: 10.1002/cbic.201402064
• Languages: English
• ISSNs: 1439-4227

Abstract

<abstract abstract-type="main" xml:lang="en"> <title>Abstract</title> <p>Translocase MraY is the site of action of lysis protein E from bacteriophage ϕX174. Previous genetic studies have shown that mutation F288L in transmembrane helix 9 of <italic>E. coli</italic> MraY confers resistance to protein E. Construction of a helical wheel model for transmembrane helix 9 of MraY and the transmembrane domain of protein E enabled the identification of an Arg‐Trp‐x‐x‐Trp (RWxxW) motif in protein E that might interact with Phe288 of MraY and the neighbouring Glu287. This motif is also found in a number of cationic antimicrobial peptide sequences. Synthetic dipeptides and pentapeptides based on the RWxxW consensus sequence showed inhibition of particulate <italic>E. coli</italic> MraY activity (IC<sub>50</sub> 200–600 μ<sc>M</sc>), and demonstrated antimicrobial activity against <italic>E. coli</italic> (MIC 31–125 μg mL<sup>−1</sup>). Cationic antimicrobial peptides at a concentration of 100 μg mL<sup>−1</sup> containing Arg‐Trp sequences also showed 30–60 % inhibition of <italic>E. coli</italic> MraY activity. Assay of the synthetic peptide inhibitors against recombinant MraY enzymes from <italic>Bacillus subtilis</italic>, <italic>Pseudomonas aeruginosa</italic>, and <italic>Micrococcus flavus</italic> (all of which lack Phe288) showed reduced levels of enzyme inhibition, and assay against recombinant <italic>E. coli</italic> MraY F288L and an E287A mutant demonstrated either reduced or no detectable enzyme inhibition, thus indicating that these peptides interact at this site. The MIC of Arg‐Trp‐octyl ester against <italic>E. coli</italic> was increased eightfold by overexpression of <italic>mraY</italic>, and was further increased by overexpression of the <italic>mraY</italic> mutant F288L, also consistent with inhibition at the RWxxW site. As this site is on the exterior face of the cytoplasmic membrane, it constitutes a potential new site for antimicrobial action, and provides a new cellular target for cationic antimicrobial peptides.</p> </abstract>