Structure of the LpxC deacetylase with a bound substrate-analog inhibitor

Abstract

The zinc-dependent UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) catalyzes the first committed step in the biosynthesis of lipid A, the hydrophobic anchor of lipopolysaccharide (LPS) that constitutes the outermost monolayer of Gram-negative bacteria. As LpxC is crucial for the survival of Gram-negative organisms and has no sequence homology to known mammalian deacetylases or amidases, it is an excellent target for the design of new antibiotics. The solution structure of LpxC from Aquifex aeolicus in complex with a substrate-analog inhibitor, TU-514, reveals a novel alpha/beta fold, a unique zinc-binding motif and a hydrophobic passage that captures the acyl chain of the inhibitor. On the basis of biochemical and structural studies, we propose a catalytic mechanism for LpxC, suggest a model for substrate binding and provide evidence that mobility and dynamics in structural motifs close to the active site have key roles in the capture of the substrate.

Figure 1

LpxC reaction and structure of TU-514. Functional groups found on substrate but not on TU-514 are in red; those unique to TU-514 are in blue. Kdo, 3-deoxy-D-manno-octulosonic acid; ACP, acyl carrier protein. Carbons of TU-514 hexose ring are numbered as with an equivalent sugar, from 1 to 6. Protons at position 1 are distinguished between axial (1a) and equatorial (1e). The α-methylene of the hydroxamate group is labeled Z. Acyl chain is numbered from 1 (carbonyl carbon) to 14 (terminal methyl). Atom s in the acyl chain are labeled (A), those from the glucose-like ring (G) and those in the hydroxamate (H).

Figure 2

Solution structure of LpxC in complex with TU-514. (a) Stereo view of backbone traces from the 15 final structures of the com plex, colored by secondary structure (α-helices, red; β-strands, blue; loop regions, gray). Residues 271–28 2 are disordered and are not shown. TU-514 is shown in magenta. Zinc ion locations from these structures are superimposed in a space-filling representation (coral). (b) Ribbon representation of structure colored by domain or domain insert. Linkers between domains and between domains and their inserts are colored gray. TU-514 is shown as a space-filling model with CPK coloring (carbon, black; hydrogen, white; oxygen, red; nitrogen, blue); zinc ion is shown as a space-filling model beside it. (c) Sequences of the LpxC enzymes from A. aeolicus and E. coli are aligned, with zinc-coordinating residues in magenta and conserved residues important for catalysis in orange. Secondary structure of A. aeolicus LpxC is indicated above the sequence, colored as in b, and the relevant residues are boxed in. Panels a and b were generated with MolMol.

Figure 3

The TU-514 acyl chain binds in a hydrophobic passage. (a) Surface of complex with TU-514 protruding. Residues that interact with the acyl chain of TU-514 are colored yellow and labeled, except for Thr179, which is at the bottom of the passage underneath the acyl chain. (b) Sample intermolecular NOE crosspeaks between TU-514 and LpxC. Strips corresponding to particular resonances of LpxC are shown, and the atoms on TU-514 correlated by each crosspeak are indicated. The chemical shift of each diagonal peak is labeled below that strip. TU-514 atom nomenclature is described in Figure 1 legend. Panel a was generated with PyMOL (DeLano Scientific).

Figure 4

Active-site structure and proposed catalytic mechanism. (a) Active site of LpxC with TU-514 bound. Coordination between His74, His226 and Asp230 and the zinc ion is shown with dashed lines (magenta). (b) Proposed mechanism of LpxC showing model of reaction transition state. Water activation by His253 and nucleophilic attack of the carbonyl carbon by the activated water molecule are shown with red arrows. Dashed lines indicate partial bonding between water and the substrate (yellow), coordination of ligands to zinc (magenta) and salt bridges between the oxyanion and Lys227 and between His253 and Asp234 (blue). Ribbons are colored by domain as in Figure 2. Generated with MolMol.

Figure 5

Model for native substrate binding. (a) Overlay of the five lowest-energy calculated structures (gold). Position of TU-514 is shown in magenta. LpxC ribbon diagram is colored by domain as in Figure 2. (b) Model of His19 mediating the negative charges on the phosphates of the substrate. Generated with MolMol.

Figure 6

Perturbations and missing resonances of free LpxC mapped to the structure of the LpxC-TU-514 complex. Chemical shift perturbations are calculated as (δ_H² + 0.2δ_N²)^1/2. Residues with perturbations are shown with colors on a gradient between deep blue (little change) and green (largest change); missing resonances in the absence of TU-514 are colored red. Generated with MolMol.