The Essential Escherichia coli Apolipoprotein N-Acyltransferase (Lnt) Exists as an Extracytoplasmic Thioester Acyl-Enzyme Intermediate

Abstract

Escherichia coli apolipoprotein N-acyltransferase (Lnt) transfers an acyl group from sn-1-glycerolphospholipid to the free α-amino group of the N-terminal cysteine of apolipoproteins, resulting in mature triacylated lipoprotein. Here we report that the Lnt reaction proceeds through an acyl enzyme intermediate in which a palmitoyl group forms a thioester bond with the thiol of active site residue C387 that was cleaved by neutral hydroxylamine. Lnt(C387S) also formed a fatty acyl intermediate that was resistant to neutral hydroxylamine treatment, consistent with formation of an oxygen-ester linkage. Lnt(C387A) did not form an acyl enzyme intermediate and, like Lnt(C387S), did not have any detectable Lnt activity, indicating that acylation can not occur at other positions in the catalytic domain. The existence of this thioacyl-enzyme intermediate allowed us to determine whether essential residues in the catalytic domain of Lnt affect the first step of the reaction, the formation of the acyl enzyme intermediate, or the second step in which the acyl chain is transferred to apolipoprotein substrate. In the catalytic triad, E267 is required for the formation of the acyl-enzyme intermediate, indicating its role in enhancing the nucleophilicity of C387. E343 is also involved in the first step but is not in close proximity to the active site. W237, Y388 and E389 play a role in the second step of the reaction since acyl-Lnt is formed but N-acylation does not occur. The data presented allow discrimination between the functions of essential Lnt residues in catalytic activity and substrate recognition.

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

“…Previous studies have demonstrated that mutating the active-site cysteine of Lnt to a serine leads to the formation of an acyl-enzyme intermediate that is resistant to reducing agents (25). Such mutants are also unable to efficiently resolve the acyl enzyme intermediate in the second step of N-acylation, likely owing to the substantially higher pK a of the serine side chain, which makes it a poor leaving group compared with the cysteine in the wild-type enzyme.…”

“…4D). Additionally, it is wellestablished that W237 is essential for Lnt activity (24,25,27), although the reason behind this requirement has been elusive. Based on our structure, we therefore propose this structural element to be a potential phosphate-recognition site.…”

“…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).…”

“…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). Other residues have been reported to be essential for the activity of Lnt, however their function remains unclear due to a lack of an atomic structure (24,25,27).…”

“…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). Other residues have been reported to be essential for the activity of Lnt, however their function remains unclear due to a lack of an atomic structure (24,25,27). Despite considerable biochemical insights, the molecular mechanisms that underlie lipid binding and apolipoprotein N-acylation by Lnt remain unknown.…”