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J Biol Chem. 2002 Jul 19;277(29):25947-56. Epub 2002 May 8.

Accumulation of the lipid A precursor UDP-2,3-diacylglucosamine in an Escherichia coli mutant lacking the lpxH gene.

Author information

  • 1Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.

Abstract

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 degrees C but loses viability at 44 degrees C, demonstrating that lpxH is essential. CDP-diglyceride hydrolase (Cdh) catalyzes the same reaction as LpxH in vitro but is non-essential 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 degrees 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 degrees 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 degrees 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.

PMID:
12000771
[PubMed - indexed for MEDLINE]
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