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

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.

SUBMITTER: Rutten L

PROVIDER: S-EPMC2644146 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

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

Rutten Lucy L Mannie Jean-Paul B A JP Stead Christopher M CM Raetz Christian R H CR Reynolds C Michael CM Bonvin Alexandre M J J AM Tommassen Jan P JP Egmond Maarten R MR Trent M Stephen MS Gros Piet P

Proceedings of the National Academy of Sciences of the United States of America 20090127 6

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 th ...[more]

PMID: 19174515

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Project description:BackgroundSalmonella enterica serotype Typhimurium is a nontyphoidal and common foodborne pathogen that causes serious threat to humans. There is no licensed vaccine to prevent the nontyphoid bacterial infection caused by S. Typhimurium.MethodsTo develop conjugate vaccines, the bacterial lipid-A free lipopolysaccharide (LFPS) is prepared as the immunogen and used to synthesize the LFPS-linker-protein conjugates 6a-9b. The designed bifunctional linkers 1-5 comprising either an o-phenylenediamine or amine moiety are specifically attached to the exposed 3-deoxy-D-manno-octulosonic acid (Kdo), an α-ketoacid saccharide of LFPS, via condensation reaction or decarboxylative amidation. In addition to bovine serum albumin and ovalbumin, the S. Typhimurium flagellin (FliC) is also used as a self-adjuvanting protein carrier.ResultsThe synthesized conjugate vaccines are characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and fast performance liquid chromatography (FPLC), and their contents of polysaccharides and protein are determined by phenol-sulfuric acid assay and bicinchoninic acid assay, respectively. Enzyme-linked immunosorbent assay (ELISA) shows that immunization of mouse with the LFPS-linker-protein vaccines at a dosage of 2.5 μg is sufficient to elicit serum immunoglobulin G (IgG) specific to S. Typhimurium lipopolysaccharide (LPS). The straight-chain amide linkers in conjugates 7a-9b do not interfere with the desired immune response. Vaccines 7a and 7b derived from either unfractionated LFPS or the high-mass portion show equal efficacy in induction of IgG antibodies. The challenge experiments are performed by oral gavage of S. Typhimurium pathogen, and vaccine 7c having FliC as the self-adjuvanting protein carrier exhibits a high vaccine efficacy of 74% with 80% mice survival rate at day 28 post the pathogen challenge.ConclusionsThis study demonstrates that lipid-A free lipopolysaccharide prepared from Gram-negative bacteria is an appropriate immunogen, in which the exposed Kdo is connected to bifunctional linkers to form conjugate vaccines. The decarboxylative amidation of Kdo is a novel and useful method to construct a relatively robust and low immunogenic straight-chain amide linkage. The vaccine efficacy is enhanced by using bacterial flagellin as the self-adjuvanting carrier protein.

Dataset Information

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

Publications

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

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