Enzymatic Synthesis of Lipid A Molecules with Four Amide-linked Acyl Chains

Abstract

LpxA of Escherichia coli catalyzes the acylation of the glucosamine 3-OH group of UDP-GlcNAc, using R-3-hydroxymyristoyl-acyl carrier protein (ACP) as the donor substrate. We now demonstrate that LpxA in cell extracts of Mesorhizobium loti and Leptospira interrogans, The mechanism of L. interrogans LpxA appears to be similar to that of E. coli LpxA, given that the essential His 125 residue of E. coli LpxA is conserved and is also required for acyltransferase activity in L. interrogans. Acidithiobacillus ferrooxidans (an organism that makes lipid A molecules containing both GlcN and GlcN3N) has an ortholog of LpxA that is selective for UDP-GlcNAc3N, but the enzyme also catalyzes the acylation of UDPGlcNAc at a slow rate. E. coli LpxA acylates UDP-GlcNAc and UDP-GlcNAc3N at comparable rates in vitro. However, UDP-GlcNAc3N is not synthesized in vivo, because E. coli lacks gnnA and gnnB. When the latter are supplied together with A. ferrooxidans lpxA, E. coli incorporates a significant amount of GlcN3N into its lipid A.

Structural basis for the acyl chain selectivity and mechanism of UDP- N -acetylglucosamine acyltransferase

“…Role of conserved LpxA residues in acyl chain selectivity and catalysis. Our structural analysis supports the previously proposed role of H125 as the catalytic base (18,32) and further identifies the functions of various conserved side chains in substrate binding during the tetrahedral transition state, as indicated. Uncertainties remain about the positioning of the phosphopantetheine arm of ACP and the role of the conserved H160 residue (Fig.…”

“…Structures of LpxA orthologues with intrinsically different acyl chain selectivity, such as the C10-selective P. aeruginosa LpxA (31) or the C12-selective Leptospira interrogans LpxA (32), might provide further insights in the acyl chain recognition mechanism. L. interrogans LpxA displays the additional interesting feature of requiring as its acceptor substrate an analogue of UDP-GlcNAc, in which an amine replaces the GlcNAc O-3 atom (32,33). E. coli LpxA utilizes both of these sugar nucleotides at comparable rates but cannot synthesize the analogue (32).…”

Lipid A Modification Systems in Gram-Negative Bacteria

“…Many bacteria (94a), including L. interrogans and Acidithiobacillus ferrooxidans, contain a dehydrogenase (GnnA) and a transaminase (GnnB) that convert UDP-GlcNAc to the analogue UDP-GlcNAc3N, in which the GlcNAc 3-OH group is replaced with an amine (Figure 4) (89,95). LpxA of L. interrogans, which is absolutely selective for a C12 chain (89,95) (Figure 4), acylates UDPGlcNAc3N but not UDP-GlcNAc (89,95).…”

“…The active site of E. coli LpxA functions as a precise hydrocarbon ruler that incorporates C14 hydroxyacyl chains two orders of magnitude faster than C12 or C16 chains (86,87), consistent with the structure of E. coli lipid A (Figure 2). In Pseudomonas aeruginosa, the LpxA ruler is reset to incorporate C10 chains (86,87), whereas in Neisseria meningitidis and Leptospira interrogans, it measures C12 chains (88,89). Strains of E. coli in which P. aeruginosa lpxA replaces E. coli lpxA synthesize hybrid lipid A molecules in which C10 acyl chains are incorporated at positions 3 and 3 ( Figure 2, red numbers).…”

A Leptospira interrogans Enzyme with Similarity to Yeast Ste14p That Methylates the 1-Phosphate Group of Lipid A

“…GnnA catalyzes the oxidation of the glucosamine 3-OH of UDP-GlcNAc, and GnnB catalyzes the subsequent transamination to form UDP-GlcNAc3N. LpxA from L. interrogans is absolutely specific for UDP-GlcNAc3N versus UDP-GlcNAc (19). E. coli LpxA uses both UDP-GlcNAc and UDP-GlcNAc3N in vitro, but it cannot synthesize the latter, because it lacks the gnnA and gnnB genes.…”