Shortened Hydroxyacyl Chains on Lipid A of Escherichia coli Cells Expressing a Foreign UDP-N-Acetylglucosamine O-Acyltransferase

Odegaard, Timna J.; Kaltashov, Igor A.; Cotter, Robert J.; Steeghs, Liana; Ley, Peter van der; Khan, Shahid A.; Maskell, Duncan J.; Raetz, Christian R. H.

doi:10.1074/jbc.272.32.19688

Cited by 65 publications

(107 citation statements)

References 53 publications

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“…The more slowly migrating form corresponds to the trisphosphate species. The molecular weights of these lipid A species were confirmed by mass spectrometry (36). 2 Both the lipid A bisphosphate and the trisphosphate species (Fig.…”

Section: Rapid Detection Of Lipid a Molecular Species In Separated Inmentioning

confidence: 92%

“…Our assay is based on the observation that the Kdo-lipid A linkage (Fig. 2) is cleaved selectively by pH 4.5 hydrolysis at 100°C in the presence of SDS without measurable loss of ester-linked fatty acids or of the anomeric phosphate (34)(35)(36). The hydrolysis procedure can be applied directly to radioactive cells or to isolated membrane fractions from a sucrose gradient without first extracting LPS with phenol-containing solvents.…”

Section: Discussionmentioning

confidence: 99%

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Function of Escherichia coli MsbA, an Essential ABC Family Transporter, in Lipid A and Phospholipid Biosynthesis

Zhou¹,

White²,

Polissi³

et al. 1998

Journal of Biological Chemistry

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323

339

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Section: Rapid Detection Of Lipid a Molecular Species In Separated Inmentioning

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Function of Escherichia coli MsbA, an Essential ABC Family Transporter, in Lipid A and Phospholipid Biosynthesis

Zhou¹,

White²,

Polissi³

et al. 1998

Journal of Biological Chemistry

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323

339

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“…Atomic coordinates for the complex have been deposited with the Protein Data Bank (ID code 2AQ9). (53), as indicated. Peptide 920 was dissolved in DMSO and preincubated with the reaction mixture at 30°C for 3 min in the absence of enzyme at concentrations ranging from 1 nM to 10,000 nM.…”

Section: Methodsmentioning

confidence: 99%

“…Reactions were terminated by spotting 1-l portions onto a silica TLC plate (53). The spots were air dried for 10 min before developing in chloroform͞methanol͞water͞acetic acid (25:15:4:2, vol͞vol).…”

Section: Methodsmentioning

confidence: 99%

Structure of UDP-N-acetylglucosamine acyltransferase with a bound antibacterial pentadecapeptide

Williams

Immormino

Gewirth

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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UDP-GlcNAc acyltransferase (LpxA) catalyzes the first step of lipid A biosynthesis, the transfer of the R-3-hydroxyacyl chain from R-3-hydroxyacyl acyl carrier protein (ACP) to the glucosamine 3-OH group of UDP-GlcNAc. LpxA is essential for the growth of Escherichia coli and related Gram-negative bacteria. The crystal structure of the E. coli LpxA homotrimer, determined previously at 2.6 Å in the absence of substrates or inhibitors, revealed that LpxA contains an unusual, left-handed parallel ␤-helix fold. We now present the crystal structure at 1.8 Å resolution of E. coli LpxA in a complex with a pentadecapeptide, peptide 920. Three peptides, each of which adopts a ␤-hairpin conformation, are bound per LpxA trimer. The peptides are located at the interfaces of adjacent subunits in the vicinity of the three active sites. Each peptide interacts with residues from both adjacent subunits. Peptide 920 is a potent inhibitor of E. coli LpxA (Ki ‫؍‬ 50 nM). It is competitive with respect to acyl-ACP but not UDP-GlcNAc. The compact ␤-turn structure of peptide 920 bound to LpxA may open previously uncharacterized approaches to the rational design of LpxA inhibitors with antibiotic activity.antibiotic ͉ crystal structure ͉ Escherichia coli ͉ inhibitor ͉ outer membrane L ipopolysaccharide constitutes the outer monolayer of the outer membrane in Gram-negative bacteria (1-3). Lipopolysaccharide maintains the integrity of the outer membrane, which functions as a barrier to molecules Ͼ500 Da (4). Lipid A (endotoxin) is the hydrophobic moiety that anchors lipopolysaccharide into the outer membrane (1, 2). It is a potent activator of the human innate immune system via Toll-like receptor 4 (TLR4) (5-7). The minimal lipopolysaccharide required for growth usually consists of two 3-deoxy-D-manno-octulosonic acid (Kdo) residues attached to lipid A ( Fig. 1; ref. 2). Because lipid A biosynthetic enzymes have no mammalian counterparts, they are attractive targets for the design of new antibiotics (10, 11).Kdo 2 -lipid A is synthesized by a conserved pathway consisting of nine enzymes (1, 2). LpxA catalyzes the first step (Fig. 1), the thermodynamically unfavorable 3-O-acylation of UDP-GlcNAc (K eq ϭ 0.01) (12-14). The crystal structure of Escherichia coli LpxA, previously determined at 2.6 Å (15), revealed that the enzyme is a homotrimer. It adopts a distinctive, left-handed parallel ␤-helix fold ( Fig. 2 A and B), which is specified by the presence of 24 complete and six partial hexad repeats (15). Three contiguous hexad repeats (18-aa residues) fold into one coil of the ␤-helix (Fig. 2 A and B). Many other bacterial acyl and acetyl transferases later were shown to possess this fold (17)(18)(19)(20)(21)(22).Structures of LpxA have not been solved in the presence of substrates or inhibitors. Mutagenesis suggests that the active site is located in a large cleft between adjacent subunits (8, 23). This region contains several conserved basic residues (K76, H122, H125, H144, H160, and R204) (23). H125 (Fig. 2 C and D) may be the catalytic ...

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“…The lpxH gene was cloned into the low copy vector pNGH1-amp (23,28) to yield pKJB6. PCR was employed to amplify lpxH from W3110 chromosomal DNA.…”

Section: Preparation Of Lipid Substrates and Assay Of Lpxh-udp-23-dimentioning

confidence: 99%

Accumulation of the Lipid A Precursor UDP-2,3-diacylglucosamine in an Escherichia coli Mutant Lacking the lpxH Gene

Babinski¹,

Kanjilal

Raetz

2002

Journal of Biological Chemistry

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The lpxH gene encodes the UDP-2,3-diacylglucosamine-specific pyrophosphatase that catalyzes the fourth step of lipid A biosynthesis in Escherichia coli. To confirm the function of lpxH, we constructed KB21/ pKJB5. This strain contains a kanamycin insertion element in the chromosomal copy of lpxH, complemented by plasmid pKJB5, which is temperature-sensitive for replication and harbors lpxH ؉ . KB21/pKJB5 grows at 30°C but loses viability at 44°C, demonstrating that lpxH is essential. CDP-diglyceride hydrolase (Cdh) catalyzes the same reaction as LpxH in vitro but is nonessential and cannot compensate for the absence of LpxH. The presence of Cdh in cell extracts interferes with the LpxH assay. We therefore constructed KB25/ pKJB5, which contains both an in-frame deletion of cdh and a kanamycin insertion mutation in lpxH, covered by pKJB5. When KB25/pKJB5 cells are grown at 44°C, viability is lost, and all in vitro LpxH activity is eliminated. A lipid migrating with synthetic UDP-2,3-diacylglucosamine accumulates in KB25/pKJB5 following loss of the covering plasmid at 44°C. This material was converted to the expected products, 2,3-diacylglucosamine 1-phosphate and UMP, by LpxH. Pseudomonas aeruginosa contains two proteins with sequence similarity to E. coli LpxH. The more homologous protein catalyzes UDP-2,3-diacylglucosamine hydrolysis in vitro. The corresponding gene complements KB25/pKJB5 at 44°C, but the less homologous gene does not. The accumulation of UDP-2,3-diacylglucosamine in our lpxH mutant is consistent with the observation that the lipid A disaccharide synthase LpxB, the next enzyme in the pathway, cannot condense two UDP-2,3-diacylglucosamine molecules, but instead utilizes UDP-2,3-diacylglucosamine as its donor and 2,3-diacylglucosamine 1-phosphate as its acceptor.

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Shortened Hydroxyacyl Chains on Lipid A of Escherichia coli Cells Expressing a Foreign UDP-N-Acetylglucosamine O-Acyltransferase

Cited by 65 publications

References 53 publications

Function of Escherichia coli MsbA, an Essential ABC Family Transporter, in Lipid A and Phospholipid Biosynthesis

Function of Escherichia coli MsbA, an Essential ABC Family Transporter, in Lipid A and Phospholipid Biosynthesis

Structure of UDP-N-acetylglucosamine acyltransferase with a bound antibacterial pentadecapeptide

Accumulation of the Lipid A Precursor UDP-2,3-diacylglucosamine in an Escherichia coli Mutant Lacking the lpxH Gene

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