Cloning, expression, and purification of UDP-3-O-acyl-GlcNAc deacetylase from Pseudomonas aeruginosa: a metalloamidase of the lipid A biosynthesis pathway.
The lpxC (envA) gene of Escherichia coli encodes UDP-3-O-acyl-GlcNAc deacetylase, the second and committed step of lipopolysaccharide biosynthesis. Although present in all gram-negative bacteria examined, the deacetylase from E. coli is the only example of this enzyme that has been expressed and purified. In order to examine other variants of this protein, we cloned the Pseudomonas aeruginosa deacetylase structural gene from a lambda library as a 5.1-kb EcoRI fragment. The LpxC reading frame encodes an inferred protein of 33,435 Da that is highly homologous to the E. coli protein and that possesses a nearly identical hydropathy profile. In order to verify function, we subcloned the P. aeruginosa lpxC gene into the T7-based expression vector pET11a. Upon induction at 30 degrees C, this construct yielded active protein to approximately 18% of the soluble fraction. We devised a novel, rapid, and reproducible assay for the deacetylase which facilitated purification of the enzyme in three steps. The purified recombinant protein was found to be highly sensitive to EDTA yet was reactivated by the addition of excess heavy metal, as was the case for crude extracts of P. aeruginosa. In contrast, deacetylase activity in crude extracts of E. coli was insensitive to EDTA, and the extracts of the envA1 mutant were sensitive in a time-dependent manner. The lpxC gene has no significant homology with amidase signature sequences. Therefore, we assign this protein to the metalloamidase family as a member with a novel structure.
Hyland
S A
SA
Department of Enzymology, Merck Research Laboratories, Rahway, New Jersey 07065, USA.
Eveland
S S
SS
Anderson
M S
MS
D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D003001:Cloning, Molecular; D004492:Edetic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D008050:Lipid A / Q000096:biosynthesis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature; D015032:Zinc / Q000494:pharmacology
D002413:Cations, Divalent; D008050:Lipid A; D011994:Recombinant Proteins; D004492:Edetic Acid; D000581:Amidohydrolases; C097307:UDP-3-O-acyl-N-acetylglucosamine deacetylase; D015032:Zinc