Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 Mar;179(6):2029–2037. doi: 10.1128/jb.179.6.2029-2037.1997

Cloning, expression, and purification of UDP-3-O-acyl-GlcNAc deacetylase from Pseudomonas aeruginosa: a metalloamidase of the lipid A biosynthesis pathway.

S A Hyland 1, S S Eveland 1, M S Anderson 1
PMCID: PMC178929  PMID: 9068651

Abstract

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.

Full Text

The Full Text of this article is available as a PDF (520.0 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anderson M. S., Bulawa C. E., Raetz C. R. The biosynthesis of gram-negative endotoxin. Formation of lipid A precursors from UDP-GlcNAc in extracts of Escherichia coli. J Biol Chem. 1985 Dec 15;260(29):15536–15541. [PubMed] [Google Scholar]
  2. Anderson M. S., Bull H. G., Galloway S. M., Kelly T. M., Mohan S., Radika K., Raetz C. R. UDP-N-acetylglucosamine acyltransferase of Escherichia coli. The first step of endotoxin biosynthesis is thermodynamically unfavorable. J Biol Chem. 1993 Sep 15;268(26):19858–19865. [PubMed] [Google Scholar]
  3. Beall B., Lutkenhaus J. Sequence analysis, transcriptional organization, and insertional mutagenesis of the envA gene of Escherichia coli. J Bacteriol. 1987 Dec;169(12):5408–5415. doi: 10.1128/jb.169.12.5408-5415.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bulawa C. E., Raetz C. R. Isolation and characterization of Escherichia coli strains defective in CDP-diglyceride hydrolase. J Biol Chem. 1984 Sep 25;259(18):11257–11264. [PubMed] [Google Scholar]
  5. Bull H. G., Thornberry N. A., Cordes E. H. Purification of angiotensin-converting enzyme from rabbit lung and human plasma by affinity chromatography. J Biol Chem. 1985 Mar 10;260(5):2963–2972. [PubMed] [Google Scholar]
  6. Ellis K. J., Morrison J. F. Buffers of constant ionic strength for studying pH-dependent processes. Methods Enzymol. 1982;87:405–426. doi: 10.1016/s0076-6879(82)87025-0. [DOI] [PubMed] [Google Scholar]
  7. Feng D. F., Doolittle R. F. Progressive sequence alignment as a prerequisite to correct phylogenetic trees. J Mol Evol. 1987;25(4):351–360. doi: 10.1007/BF02603120. [DOI] [PubMed] [Google Scholar]
  8. Fleischmann R. D., Adams M. D., White O., Clayton R. A., Kirkness E. F., Kerlavage A. R., Bult C. J., Tomb J. F., Dougherty B. A., Merrick J. M. Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science. 1995 Jul 28;269(5223):496–512. doi: 10.1126/science.7542800. [DOI] [PubMed] [Google Scholar]
  9. Goldberg J. B., Ohman D. E. Cloning and expression in Pseudomonas aeruginosa of a gene involved in the production of alginate. J Bacteriol. 1984 Jun;158(3):1115–1121. doi: 10.1128/jb.158.3.1115-1121.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gribskov M., Lüthy R., Eisenberg D. Profile analysis. Methods Enzymol. 1990;183:146–159. doi: 10.1016/0076-6879(90)83011-w. [DOI] [PubMed] [Google Scholar]
  11. Gribskov M., McLachlan A. D., Eisenberg D. Profile analysis: detection of distantly related proteins. Proc Natl Acad Sci U S A. 1987 Jul;84(13):4355–4358. doi: 10.1073/pnas.84.13.4355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Huang T. L., Dredar S. A., Manneh V. A., Blankenship J. W., Fries D. S. Inhibition of N8-acetylspermidine deacetylase by active-site-directed metal coordinating inhibitors. J Med Chem. 1992 Jun 26;35(13):2414–2418. doi: 10.1021/jm00091a009. [DOI] [PubMed] [Google Scholar]
  13. Kaplan A. P., Bartlett P. A. Synthesis and evaluation of an inhibitor of carboxypeptidase A with a Ki value in the femtomolar range. Biochemistry. 1991 Aug 20;30(33):8165–8170. doi: 10.1021/bi00247a011. [DOI] [PubMed] [Google Scholar]
  14. Lenney J. F., Baslow M. H., Sugiyama G. H. Similarity of tuna N-acetylhistidine deacetylase and cod fish anserinase. Comp Biochem Physiol B. 1978;61(2):253–258. doi: 10.1016/0305-0491(78)90171-2. [DOI] [PubMed] [Google Scholar]
  15. Onishi H. R., Pelak B. A., Gerckens L. S., Silver L. L., Kahan F. M., Chen M. H., Patchett A. A., Galloway S. M., Hyland S. A., Anderson M. S. Antibacterial agents that inhibit lipid A biosynthesis. Science. 1996 Nov 8;274(5289):980–982. doi: 10.1126/science.274.5289.980. [DOI] [PubMed] [Google Scholar]
  16. Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J., Klenk D. C. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct;150(1):76–85. doi: 10.1016/0003-2697(85)90442-7. [DOI] [PubMed] [Google Scholar]
  17. Sorensen P. G., Lutkenhaus J., Young K., Eveland S. S., Anderson M. S., Raetz C. R. Regulation of UDP-3-O-[R-3-hydroxymyristoyl]-N-acetylglucosamine deacetylase in Escherichia coli. The second enzymatic step of lipid a biosynthesis. J Biol Chem. 1996 Oct 18;271(42):25898–25905. doi: 10.1074/jbc.271.42.25898. [DOI] [PubMed] [Google Scholar]
  18. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  19. Taunton J., Hassig C. A., Schreiber S. L. A mammalian histone deacetylase related to the yeast transcriptional regulator Rpd3p. Science. 1996 Apr 19;272(5260):408–411. doi: 10.1126/science.272.5260.408. [DOI] [PubMed] [Google Scholar]
  20. Williamson J. M., Anderson M. S., Raetz C. R. Acyl-acyl carrier protein specificity of UDP-GlcNAc acyltransferases from gram-negative bacteria: relationship to lipid A structure. J Bacteriol. 1991 Jun;173(11):3591–3596. doi: 10.1128/jb.173.11.3591-3596.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Yoshida M., Kijima M., Akita M., Beppu T. Potent and specific inhibition of mammalian histone deacetylase both in vivo and in vitro by trichostatin A. J Biol Chem. 1990 Oct 5;265(28):17174–17179. [PubMed] [Google Scholar]
  22. Young K., Silver L. L., Bramhill D., Cameron P., Eveland S. S., Raetz C. R., Hyland S. A., Anderson M. S. 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. 1995 Dec 22;270(51):30384–30391. doi: 10.1074/jbc.270.51.30384. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES