title: Kinetics and phospholipid specificity of apolipoprotein N-acyltransferase.
abstract: The enzyme apolipoprotein N-acyltransferase (Lnt) is an integral membrane protein that catalyzes the last step in the post-translational modification of bacterial lipoproteins. Lnt undergoes covalent modification in the presence of phospholipids resulting in a thioester acyl-enzyme intermediate. It then transfers the acyl chain to the α-amino group of the N-terminal diacylglyceryl-modified cysteine of apolipoprotein, leading to the formation of mature triacylated lipoprotein. To gain insight into the catalytic mechanism of this two-step reaction, we overproduced and purified the enzyme of Escherichia coli and studied its N-acyltransferase activity using a novel in vitro assay. The purified enzyme was fully active, as judged by its ability to form a stable thioester acyl-enzyme intermediate and N-acylate the apo-form of the murein lipoprotein Lpp in vitro. Incorporation of [(3)H]palmitate and mass spectrometry analysis demonstrated that Lnt recognized the synthetic diacylglyceryl-modified lipopeptide FSL-1 as a substrate in a mixed micelle assay. Kinetics of Lnt using phosphatidylethanolamine as an acyl donor and FSL-1 as a substrate were consistent with a ping-pong type mechanism, demonstrating slow acyl-enzyme intermediate formation and rapid N-acyl transfer to the apolipopeptide in vitro. In contrast to earlier in vitro observations, the N-acyltransferase activity was strongly affected by the phospholipid headgroup and acyl chain composition.
authors.affiliation: Institut Pasteur, CNRS URA 2172, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, 91198 Gif-sur-Yvette Cedex, France.
authors.forename: Falk
authors.initials: F
authors.lastname: Hillmann
authors.forename: Manuela
authors.initials: M
authors.lastname: Argentini
authors.forename: Nienke
authors.initials: N
authors.lastname: Buddelmeijer
mesh_terms: D000217:Acyltransferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008074:Lipoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity / Q000502:physiology
chemical_list: D029968:Escherichia coli Proteins; D008074:Lipoproteins; D010743:Phospholipids; D011994:Recombinant Proteins; D000217:Acyltransferases; C068902:apolipoprotein N-acyltransferase