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

Abstract

UDP-N-acetylglucosamine (UDP-GlcNAc) acyltransferase (LpxA) catalyzes the first step of lipid A biosynthesis, the reversible transfer of the R-3-hydroxyacyl chain from R-3-hydroxyacyl acyl carrier protein to the glucosamine 3-OH group of UDP-GlcNAc. Escherichia coli LpxA is highly selective for R-3-hydroxymyristate. The crystal structure of the E. coli LpxA homotrimer, determined previously in the absence of lipid substrates or products, revealed that LpxA contains an unusual, left-handed parallel beta-helix fold. We have now solved the crystal structures of E. coli LpxA with the bound product UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc at a resolution of 1.74 A and with bound UDP-3-O-(R-3-hydroxydecanoyl)-GlcNAc at 1.85 A. The structures of these complexes are consistent with the catalytic mechanism deduced by mutagenesis and with a recent 3.0-A structure of LpxA with bound UDP-GlcNAc. Our structures show how LpxA selects for 14-carbon R-3-hydroxyacyl chains and reveal two modes of UDP binding.

Fig. 1.

Functions of LpxA and LpxC in lipid A biosynthesis. LpxA catalyzes the first step, the acylation of UDP-GlcNAc (2, 4). This is a thermodynamically unfavorable reaction; therefore, LpxC catalyzes the committed step of the pathway (8).

Fig. 2.

Structural models of two LpxA/product complexes. (A) Side view of LpxA (ribbon diagram) with UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc (space filling model) at a resolution of 1.74 Å. Individual monomers of the LpxA homotrimer are colored green, magenta, and blue. The UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc product binds within the active site region located between adjacent subunits, as anticipated from site-directed mutagenesis studies (18). In the space-filling model of the ligand, carbon is yellow, nitrogen is blue, oxygen is red, and phosphorus is orange. (B) The top-down view demonstrates the threefold symmetry of the bound ligand. (C) The side view of LpxA (ribbon diagram) with bound UDP-3-O-(R-3-hydroxydecanoyl)-GlcNAc (space-filling model) at a resolution of 1.85 Å. The color scheme is similar to that described in A, except that the carbon atoms of the R-3-hydroxydecanoyl chain are gray. (D) Top-down view of the LpxA/UDP-3-O-(R-3-hydroxydecanoyl)-GlcNAc complex.

Fig. 3.

Stereoviews of the electron densities of the bound ligands. (A) Stereoview of the final 2F_o − F_c electron density maps surrounding UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc contoured at 1.2 σ. (B) Stereoview of the final 2F_o − F_c electron density maps surrounding UDP-3-O-(R-3-hydroxydecanoyl)-GlcNAc contoured at 1.2 σ. The color scheme for the stick models of these ligands is the same as in Fig. 2.

Fig. 4.

Positioning of conserved residues within the active site of E. coli LpxA. (A) Stereoview of the interactions between residues in the active site of LpxA and UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc. The color scheme is the same as in Fig. 2. Water molecules are depicted as cyan spheres. Hydrogen bonds are represented as black dashed lines. Hydrogen bonds between water molecules and the product molecule were omitted for clarity. G134, the putative oxyanion hole, is slightly too far removed from the carbonyl group of the acyl chain for H-bonding (3.32–3.35 Å), but it might be able to interact during catalysis. (B) Stereoview of the interactions between LpxA and UDP-3-O-(R-3-hydroxydecanoyl)-GlcNAc. The color scheme is the same as in Fig. 2. The most significant differences are in the binding and orientation of the UDP moiety of the two ligands. In both structures, Q73 makes an additional hydrogen bond (not shown for clarity) via its backbone nitrogen atom to the single water molecule that is hydrogen-bonded to the R-3-OH moiety of the ligand.

Fig. 5.

Alternative conformations for the UDP moiety in LpxA/ligand complexes. (A) Superimposition of the ligands in the LpxA/UDP-3-O-(R-3-hydroxydecanoyl)-GlcNAc complex and the LpxA/UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc complex highlights the similarities and differences in the binding of the ligands. The positions of the atoms within the GlcNAc residue, the first four carbon and oxygen atoms of the acyl chain, and the β-phosphate group are positioned identically. However, the α-phosphate, the ribose ring, and the uracil moiety assume an entirely different orientation, which nevertheless permits the uracil moiety to hydrogen-bond to the side chain of N198 in both instances (Fig. 4). The color scheme is the same as in Fig. 2. (B) Superimposition of the ligands in the LpxA/UDP-3-O-(R-3-hydroxydecanoyl)-GlcNAc complex and the 3-Å LpxA/UDP-GlcNAc complex (27). Carbons of the latter ligand are green. The conformations of the GlcNAc and UDP moieties are virtually identical for these two complexes, showing that the presence of the longer R-3-hydroxymyristoyl chain exerts subtle effects on UDP binding.

Fig. 6.

Positioning of the end of the R-3-hydroxymyristoyl chain near G173. This view displays one of the three active sites of LpxA with bound UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc. The carbon atoms of the ligand (sticks with dots) are yellow, and the terminal methyl group (C14) is near the top, in the vicinity of H191. C12 of the R-3-hydroxymyristoyl chain is located in close proximity to G173. The terminal methyl group of the acyl chain of UDP-3-O-(R-3-hydroxydecanoyl)-GlcNAc (not shown) would be located between carbons 9 and 10 of UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc (Fig. 5A).

Fig. 7.

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. 6), the side chain of which is displaced by the presence of the acyl chain of the ligand. The proposed role of G143 as the oxyanion hole is inferred on the basis of the absolute conservation of this residue and its positioning near the carbonyl oxygen atoms of the ligands, as shown in Fig. 4.

Fig. 8.

Structures of the first three enzymes of the lipid A pathway with bound lipid ligands. (A) Crystal structure of E. coli LpxA with bound UDP-3-O-(R-3-hydroxydecanoyl)-GlcNAc, as determined in the present study; only one bound ligand is shown for clarity. (B) Representative NMR structure of Aquifex aeolicus LpxC with bound substrate-mimetic inhibitor TU514. The catalytic zinc atom is magenta. LpxC consists of a single polypeptide chain, colored with a gradient of colors (N terminus in blue to the C terminus in red). The acyl chain of the inhibitor is trapped within an unusual tunnel, which opens to solvent at the surface of the protein (48, 49). This structural feature explains why LpxC requires the presence of a 3-O-linked acyl chain in its substrate for activity and why LpxC is not very selective with regard to the length of that acyl chain. (C) Crystal structure of the trimeric C. trachomatis LpxD with bound UDP-GlcNAc and a free fatty acid (39), showing the similarity of the β-helix domain to that of LpxA and the location of one of the active sites. The natural LpxD acceptor substrate is UDP-3-O-(R-3-hydroxyacyl)-glucosamine. The locations of the acyl-ACP binding sites on LpxA and LpxD are unknown.