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PMID:12000771

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Citation

Babinski, KJ, Kanjilal, SJ and Raetz, CR (2002) Accumulation of the lipid A precursor UDP-2,3-diacylglucosamine in an Escherichia coli mutant lacking the lpxH gene. J. Biol. Chem. 277:25947-56

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.

Links

PubMed Online version:10.1074/jbc.M204068200

Keywords

Amino Acid Sequence; Diglycerides/metabolism; Electrophoresis, Polyacrylamide Gel; Escherichia coli/enzymology; Escherichia coli/genetics; Kanamycin; Lipid A/biosynthesis; Molecular Sequence Data; Mutagenesis, Insertional; N-Acetylglucosaminyltransferases/metabolism; Plasmids; Pseudomonas aeruginosa; Pyrophosphatases/genetics; Pyrophosphatases/metabolism; Sequence Alignment; Temperature; Uridine Diphosphate/analogs & derivatives; Uridine Diphosphate/metabolism

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

ECOLI:LPXH

located_in

GO:0031234: extrinsic component of cytoplasmic side of plasma membrane

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

ECOLI:LPXH

acts_upstream_of_or_within

GO:0009245: lipid A biosynthetic process

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete

PSEAE:LPXH

involved_in

GO:0009245: lipid A biosynthetic process

ECO:0000316: genetic interaction evidence used in manual assertion

UniProtKB:P43341

P

Seeded From UniProt

complete

PSEAE:LPXH

enables

GO:0008758: UDP-2,3-diacylglucosamine hydrolase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

ECOLI:LPXH

part_of

GO:0019897: extrinsic component of plasma membrane

ECO:0000269: experimental evidence used in manual assertion

C

Seeded From UniProt

complete

ECOLI:LPXH

enables

GO:0008758: UDP-2,3-diacylglucosamine hydrolase activity

ECO:0000315: mutant phenotype evidence used in manual assertion

F

Seeded From UniProt

complete

ECOLI:LPXH

involved_in

GO:0009245: lipid A biosynthetic process

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete


See also

References

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