Active-site architecture and catalytic mechanism of the lipid A deacylase LpxR of Salmonella typhimurium - PubMed (original) (raw)

Active-site architecture and catalytic mechanism of the lipid A deacylase LpxR of Salmonella typhimurium

Lucy Rutten et al. Proc Natl Acad Sci U S A. 2009.

Abstract

The lipid A portion of lipopolysaccharide, the major component of the outer leaflet of the outer membrane of gram-negative bacteria, is toxic to humans. Modification of lipid A by enzymes often reduces its toxicity. The outer-membrane protein LpxR from Salmonella typhimurium is a lipid A-modifying enzyme. It removes the 3'-acyloxyacyl moiety of the lipid A portion of lipopolysaccharide in a Ca(2+)-dependent manner. Here, we present the crystal structure of S. typhimurium LpxR, crystallized in the presence of zinc ions. The structure, a 12-stranded beta-barrel, reveals that the active site is located between the barrel wall and an alpha-helix formed by an extracellular loop. Based on site-directed mutagenesis and modeling of a substrate on the active site, we propose a catalytic mechanism similar to that of phospholipase A2, in which a Ca(2+) forms the oxyanion hole and a histidine activates a water molecule (or a cascade of two water molecules) that subsequently attacks the carbonyl oxygen of the scissile bond.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

LpxR-catalzyed deacylation of Kdo2–lipid A. (A) In the presence of Ca2+, the outer-membrane enzyme LpxR catalyzes the hydrolysis of the ester linkage at the 3′ position of Kdo2–lipid A, releasing the intact 3′-acyloxyacyl group. (B) Enzymatic activity of purified LpxR. Purified and refolded LpxR was assayed for 3′-_O_-deacylase activity by using Kdo2–[4′-32P]lipid A as the substrate. Membranes from E. coli K-12 strain HMS174(DE3)/pLpxR1 (0.001 mg/mL) served as the positive control (lane 2). Purified LpxR at a concentration of 0.001 and 0.0001 mg/mL served as the protein source in assays spotted in lanes 3 and 4, respectively. The no enzyme control is shown in lane 1. Assays were carried out for 30 min at 30 °C with 2.5 μM Kdo2–[4′-32P]lipid A either in the presence or absence of 5 mM CaCl2. The reaction products were separated by TLC and detected with PhosphorImager analysis.

Fig. 2.

Overall structure of LpxR. (A) Ribbon representation of LpxR. The ribbon is colored with a gradient from the N terminus in blue to the C terminus in red. Aromatic residues located at the membrane boundaries are shown as cyan sticks. Membrane boundaries are indicated by black lines. The extracellular side is located at the top of the figure, and the periplasmic side is at the bottom. Active-site residues are shown as yellow sticks. Zn2+ atoms are shown as gray spheres, and glycerol molecules are shown as sticks in green. Loops, turns, the N terminus, and the C terminus are labeled. (B) LpxR viewed from the periplasmic side of the protein. The representation of the structure is similar to that in A. Figs. 2, 3, 4, S2, and S5 were prepared with PyMOL (23).

Fig. 3.

Active site of LpxR. (A) Surface representation of LpxR in wheat color. Positions of fully conserved residues are shown in dark green. Residues that are largely conserved are shown in light green. (B) Close up of the active site of LpxR. The orientation of LpxR is identical to that in Fig. 2_A_. Zn2+ ions are shown as gray spheres, a water molecule as a red sphere, and a glycerol molecule is represented as sticks with green carbons. Oxygen atoms are colored red and nitrogen blue. Short distances, i.e., H bonds or shorter distances between noncovalently bonded atoms, are shown as blue dashed lines.

Fig. 4.

Modeling of Kdo2–lipid A into the active site of LpxR and the proposed catalytic mechanism. (A) View of Kdo2–lipid A, which is shown in sticks with yellow carbons, modeled onto LpxR. Fully conserved residues are shown as sticks in cyan. Residues that are probably involved in binding of the Kdo sugars, i.e., K67, R68, and H25 are shown as blue sticks. D11 is shown in magenta. The calcium ion is shown as a gray sphere. (B) LpxR is shown as a surface representation in green with K67, R68, H25, D11, Kdo2–lipid A, and the calcium colored as in A. (C) Closeup of the catalytic site of the modeling result. The representation is the same as in A, with the exception that the view angle is different and that polar hydrogens are shown in white. (D) Proposed catalytic mechanism for LpxR. The substrate is shown in blue, protein residues are shown in cyan, the water and calcium are shown in black. The scissile bond is shown in red. Black arrows indicate the movement of the electron pairs.

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Active-site architecture and catalytic mechanism of the lipid A deacylase LpxR of Salmonella typhimurium - PubMed (original) (raw)