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 posttranslational 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 ␣-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.

Structural insights into lipoprotein N-acylation by Escherichia coli apolipoprotein N-acyltransferase

“…Similar to previous studies, these data suggested that wildtype Lnt functions in two steps via a ping-pong mechanism ( Fig. 1E) (26). The kinetics of wild-type Lnt reacting with PE in this assay are in line with results reported previously, with a k cat /K m value of 674·M −1 ·s −1 (26).…”

“…A general two-step ping-pong mechanism for N-acylation by Lnt has been proposed in which a thioester acyl-enzyme intermediate is first formed at the catalytic cysteine through nucleophilic attack on the carbonyl of the sn-1-glycerophospholipid of PE, resulting in S-palmitoylated Lnt. In the second step, the acyl-enzyme intermediate is resolved by nucleophilic attack by the α-amino group of an apolipoprotein, resulting in a mature triacylated lipoprotein (24)(25)(26). Lnt has been shown to exist primarily in the acylated form in vivo, priming it for the more efficient second step of the N-acylation reaction (25).…”

Chlamydia trachomatis Scavenges Host Fatty Acids for Phospholipid Synthesis via an Acyl-Acyl Carrier Protein Synthetase

“…In E. coli, the enzyme functions in a salvage pathway to recycle 2-acyl-GPE formed during the maturation of lipoproteins. Topologically, prolipoproteins are secreted through the inner membrane, and the subsequent processing steps, proteolytic cleavage (18) (Lgt), transfer of the diacylglycerol (19) (Lsp), and N-acylation (20) (Lnt), are located on the outer aspect of the inner membrane. The 2-acyl-GPE cycle is initiated by the transfer of a fatty acid at the 1-position of PE to the amino terminus of the lipoprotein by Lnt (21).…”

N -Acylation of Lipoproteins: Not When Sour

“…Lnt of E. coli has specificity for both the acyl chain length and composition of fatty acids; its preferred substrate is PtdEtn containing a saturated fatty acid (palmitate or C16:0) on sn-1 and an unsaturated fatty acid (oleic acid or C18:1) on sn-2 (3). The sn-1 acyl is transferred to the ␣-amino group of apolipoprotein, but the sn-2 acyl moiety plays an important role (3,4). One possibility is that, because the phospholipid substrate for Lnt changes during the stationary phase, lipoproteins can no longer be N acylated.…”

“…Signal peptidase II (Lsp) then cleaves the signal peptide, resulting in an apolipoprotein containing a free ␣-amino group. Apolipoprotein N-acyltransferase (Lnt) adds an sn-1-acyl chain derived from phosphatidylethanolamine (PtdEtn) to apolipoprotein, resulting in mature triacylated protein (3,4). The genomes of Firmicutes and Mollicutes do not encode Lnt, and, with only Lgt and Lsp present, one might assume that these bacteria possess only diacylated lipoproteins with unmodified free ␣-amino termini.…”