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The Escherichia coli Gene Encoding the UDP-2,3-diacylglucosamine Pyrophosphatase of Lipid A Biosynthesis *

Journal of Biological Chemistry, ISSN: 0021-9258, Vol: 277, Issue: 29, Page: 25937-25946
2002
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UDP-2,3-diacylglucosamine hydrolase is believed to catalyze the fourth step of lipid A biosynthesis in Escherichia coli. This reaction involves pyrophosphate bond hydrolysis of the precursor UDP-2,3-diacylglucosamine to yield 2,3-diacylglucosamine 1-phosphate and UMP. To identify the gene encoding this hydrolase, E. coli lysates generated with individual λ clones of the ordered Kohara library were assayed for overexpression of the enzyme. The sequence of λ clone 157[6E7], promoting overproduction of hydrolase activity, was examined for genes encoding hypothetical proteins of unknown function. The amino acid sequence of one such open reading frame, ybbF, is 50.5% identical to a Haemophilus influenzae hypothetical protein and is also conserved in most other Gram-negative organisms, but is absent in Gram-positives. Cell extracts prepared from cells overexpressing ybbF behind the T7 lac promoter have ∼540 times more hydrolase activity than cells with vector alone. YbbF was purified to ∼60% homogeneity, and its catalytic properties were examined. Enzymatic activity is maximal at pH 8 and is inhibited by 0.01% (or more) Triton X-100. The apparent K m for UDP-2,3-diacylglucosamine is 62 μ m. YbbF requires a diacylated substrate and does not cleave CDP-diacylglycerol. 31 P NMR studies of the UMP product generated from UDP-2,3-diacylglucosamine in the presence of 40% H 2 18 0 show that the enzyme attacks the α-phosphate group of the UDP moiety. Because ybbF encodes the specific UDP-2,3-diacylglucosamine hydrolase involved in lipid A biosynthesis, it is now designated lpxH.

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