Abstract
Lipoprotein biogenesis is essential for bacterial survival. Phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (Lgt) is an integral membrane enzyme that catalyses the first reaction of the three-step post-translational lipid modification. Deletion of the lgt gene is lethal to most Gram-negative bacteria. Here we present the crystal structures of Escherichia coli Lgt in complex with phosphatidylglycerol and the inhibitor palmitic acid at 1.9 and 1.6 Å resolution, respectively. The structures reveal the presence of two binding sites and support the previously reported structure–function relationships of Lgt. Complementation results of lgt-knockout cells with different mutant Lgt variants revealed critical residues, including Arg143 and Arg239, that are essential for diacylglyceryl transfer. Using a GFP-based in vitro assay, we correlated the activities of Lgt with structural observations. Together, the structural and biochemical data support a mechanism whereby substrate and product, lipid-modified lipobox-containing peptide, enter and leave the enzyme laterally relative to the lipid bilayer.
Highlights
Lipoprotein biogenesis is essential for bacterial survival
Lgt, which catalyses the first reaction of the pathway, recognizes the ‘lipobox’ motif (with the consensus sequence [LVI]( À 3)[ASTVI]( À 2)[GAS]( À 1)C( þ 1)) of prolipoproteins[7,8] and transfers the diacylglyceryl group from phosphatidylglycerol (PG) to the thiol group of the conserved cysteine residue in the lipobox sequence[5]
Functional studies of Escherichia coli Lgt (EcLgt) show that it is encoded by the lgt gene[9] as a 291-amino acid (33 kDa) membrane protein
Summary
Lipoprotein biogenesis is essential for bacterial survival. Phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (Lgt) is an integral membrane enzyme that catalyses the first reaction of the three-step post-translational lipid modification. The biosynthetic pathway for lipoproteins, present both in Gram-negative and Gram-positive bacteria with high GC content, consists of the following three reactions (Fig. 1a): (i) diacylglyceryl modification of pre-prolipoproteins by phosphatidylglycerol:proLipoprotein diacylGlyceryl Transferase (Lgt) to form diacylglyceryl– prolipoproteins; (ii) cleavage of signal peptide from diacylglyceryl-prolipoproteins by Lipoprotein Signal Peptidase (LspA) to form apolipoproteins; and (iii) N-acylation of apolipoproteins by an apoLipoproteiin N-acyl Transferase (Lnt), resulting in mature lipoproteins[1,5]. As this pathway has been shown to be essential for survival of Gram-negative bacteria[6], these enzymes present excellent potential targets for the generation of broad spectrum antibiotics. Mutagenesis and in vitro studies showed that the key residues essential for transacylation activity are concentrated in the periplasmic half of the central cavity, and point to a feasible mechanism of substrate recognition
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