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.
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.
Young
K
K
Department of Antibiotic Discovery and Development, Merck Research Laboratories, Rahway, New Jersey 07065, USA.
Silver
L L
LL
Bramhill
D
D
Cameron
P
P
Eveland
S S
SS
Raetz
C R
CR
Hyland
S A
SA
Anderson
M S
MS
D000483:Alleles; D000581:Amidohydrolases / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics*; D001483:Base Sequence; D002455:Cell Division / Q000235:genetics; D002463:Cell Membrane Permeability / Q000235:genetics; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D005798:Genes, Bacterial; D005838:Genotype; D008050:Lipid A / Q000096:biosynthesis*; D008074:Lipoproteins / Q000096:biosynthesis* / Q000235:genetics*; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010457:Peptidoglycan / Q000096:biosynthesis; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis; D013045:Species Specificity
D001426:Bacterial Proteins; D017931:DNA Primers; C089638:EnvA protein, Chlamydia psittaci; D008050:Lipid A; D008074:Lipoproteins; D008565:Membrane Proteins; D010457:Peptidoglycan; D011994:Recombinant Proteins; D000581:Amidohydrolases; C097307:UDP-3-O-acyl-N-acetylglucosamine deacetylase