PMID:8530464
| Citation |
Young, K, Silver, LL, Bramhill, D, Cameron, P, Eveland, SS, Raetz, CR, Hyland, SA and Anderson, MS (1995) The envA permeability/cell division gene of Escherichia coli encodes the second enzyme of lipid A biosynthesis. UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase. J. Biol. Chem. 270:30384-91 |
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| Abstract |
The envA gene of Escherichia coli has been shown previously to be essential for cell viability (Beall, B. and Lutkenhaus, J. (1987) J. Bacteriol. 169, 5408-5415), yet it encodes a protein of unknown function. Extracts of strains harboring the mutant envA1 allele display 3.5-18-fold reductions in UDP-3-O-acyl-N-acetylglucosamine deacetylase specific activity. The deacetylase is the second enzymatic step of lipid A biosynthesis. The structural gene coding for the deacetylase has not been assigned. In order to determine if the envA gene encodes the deacetylase, envA was cloned into an isopropyl-1-thio-beta-D-galactopyranoside-inducible T7-based expression system. Upon induction, a protein of the size of envA was highly overproduced, as judged by SDS-PAGE. Direct deacetylase assays of cell lysates revealed a concomitant approximately 5,000-fold overproduction of activity. Assays of the purified, overproduced EnvA protein demonstrated a further approximately 5-fold increase in specific activity. N-terminal amino acid sequencing of the purified protein showed that the first 20 amino acids matched the predicted envA nucleotide sequence. Contaminating species were present at less than 1% of the level of the EnvA protein. Thus, envA is the structural gene for UDP-3-O-acyl-GlcNAc deacetylase. Based on its function in lipid A biosynthesis, we propose the new designation lpxC for this gene. |
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| Keywords |
Alleles; Amidohydrolases/genetics; Amidohydrolases/metabolism; Bacterial Proteins/biosynthesis; Bacterial Proteins/genetics; Base Sequence; Cell Division/genetics; Cell Membrane Permeability/genetics; Cloning, Molecular; DNA Primers; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli/growth & development; Genes, Bacterial; Genotype; Lipid A/biosynthesis; Lipoproteins/biosynthesis; Lipoproteins/genetics; Membrane Proteins; Molecular Sequence Data; Mutagenesis; Peptidoglycan/biosynthesis; Plasmids; Polymerase Chain Reaction; Recombinant Proteins/biosynthesis; Species Specificity |
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