Residues located on membrane-embedded flexible loops are essential for the second step of the apolipoprotein N-acyltransferase reaction

Abstract : Apolipoprotein N-acyltransferase (Lnt) is an essential membrane-bound enzyme that catalyzes the third and last step in the post-translational modification of bacterial lipoproteins. In order to identify essential residues implicated in substrate recognition and/or binding we screened for non-functional variants of Lnt obtained by error-prone polymerase chain reaction in a complementation assay using a lnt depletion strain. Mutations included amino acid substitutions in the active site and of residues located on flexible loops in the catalytic periplasmic domain. All, but one mutation , led to the formation of the thioester acyl-enzyme intermediate and to the accumulation of apo-Lpp, suggesting that these residues are involved in the second step of the reaction. A large cytoplasmic loop contains a highly conserved region and two hydrophobic segments. Accessibility analysis to alkylating reagents of substituted cysteine residues introduced in this region demonstrated that the hydrophobic segments do not completely span the membrane. Two residues in the highly conserved cytoplasmic region were shown to be essential for Lnt function. Together, our data suggest that amino acids located on flexible cyto-plasmic and periplasmic loops, predicted to be membrane embedded, are required for efficient N-acylation of lipoproteins.
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Molecular Microbiology, Wiley, 2015, 95 (4), pp.692 - 705. 〈10.1111/mmi.12897〉
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Sébastien Gélis-Jeanvoine, Stephen Lory, Jacques Oberto, Nienke Buddelmeijer. Residues located on membrane-embedded flexible loops are essential for the second step of the apolipoprotein N-acyltransferase reaction. Molecular Microbiology, Wiley, 2015, 95 (4), pp.692 - 705. 〈10.1111/mmi.12897〉. 〈pasteur-01407474〉

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