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. 1996 Sep 3;93(18):9414–9419. doi: 10.1073/pnas.93.18.9414

Use of a designed fusion protein dissociates allosteric properties from the dodecameric state of Pseudomonas aeruginosa catabolic ornithine carbamoyltransferase.

N Mouz 1, C Tricot 1, C Ebel 1, Y Petillot 1, V Stalon 1, O Dideberg 1
PMCID: PMC38442  PMID: 8790344

Abstract

The catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa, an enzyme consisting of 12 identical 38-kDa subunits, displays allosteric properties, namely carbamoylphosphate homotropic cooperativity and heterotropic activation by AMP and other nucleoside monophosphates and inhibition by polyamines. To shed light on the effect of the oligomeric organization on the enzyme's activity and/or allosteric behavior, a hybrid ornithine carbamoyltransferase/glutathione S-transferase (OTCase-GST) molecule was constructed by fusing the 3' end of the P. aeruginosa arcB gene (OTCase) to the 5' end of the cDNA encoding Musca domestica GST by using a polyglycine encoding sequence as a linker. The fusion protein was overexpressed in Escherichia coli and purified from cell extracts by affinity chromatography, making use of the GST domain. It was found to exist as a trimer and to retain both the homotropic and heterotropic characteristic interactions of the wild-type catabolic OTCase but to a lower extent as compared with the wild-type OTCase. The dodecameric organization of catabolic P. aeruginosa OTCase may therefore be related to an enhancement of the substrate cooperativity already present in its trimers (and perhaps also to the thermostability of the enzyme).

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Selected References

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  1. Baur H., Stalon V., Falmagne P., Luethi E., Haas D. Primary and quaternary structure of the catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa. Extensive sequence homology with the anabolic ornithine carbamoyltransferases of Escherichia coli. Eur J Biochem. 1987 Jul 1;166(1):111–117. doi: 10.1111/j.1432-1033.1987.tb13489.x. [DOI] [PubMed] [Google Scholar]
  2. Baur H., Tricot C., Stalon V., Haas D. Converting catabolic ornithine carbamoyltransferase to an anabolic enzyme. J Biol Chem. 1990 Sep 5;265(25):14728–14731. [PubMed] [Google Scholar]
  3. Dirr H., Reinemer P., Huber R. X-ray crystal structures of cytosolic glutathione S-transferases. Implications for protein architecture, substrate recognition and catalytic function. Eur J Biochem. 1994 Mar 15;220(3):645–661. doi: 10.1111/j.1432-1033.1994.tb18666.x. [DOI] [PubMed] [Google Scholar]
  4. Gaboriaud C., Bissery V., Benchetrit T., Mornon J. P. Hydrophobic cluster analysis: an efficient new way to compare and analyse amino acid sequences. FEBS Lett. 1987 Nov 16;224(1):149–155. doi: 10.1016/0014-5793(87)80439-8. [DOI] [PubMed] [Google Scholar]
  5. Habig W. H., Pabst M. J., Jakoby W. B. Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J Biol Chem. 1974 Nov 25;249(22):7130–7139. [PubMed] [Google Scholar]
  6. Harpaz Y., Gerstein M., Chothia C. Volume changes on protein folding. Structure. 1994 Jul 15;2(7):641–649. doi: 10.1016/s0969-2126(00)00065-4. [DOI] [PubMed] [Google Scholar]
  7. Hass D., Evans R., Mercenier A., Simon J. P., Stalon V. Genetic and physiological characterization of Pseudomonas aeruginosa mutants affected in the catabolic ornithine carbamoyltransferase. J Bacteriol. 1979 Sep;139(3):713–720. doi: 10.1128/jb.139.3.713-720.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ji X., Zhang P., Armstrong R. N., Gilliland G. L. The three-dimensional structure of a glutathione S-transferase from the mu gene class. Structural analysis of the binary complex of isoenzyme 3-3 and glutathione at 2.2-A resolution. Biochemistry. 1992 Oct 27;31(42):10169–10184. doi: 10.1021/bi00157a004. [DOI] [PubMed] [Google Scholar]
  9. Koshland D. E., Jr, Némethy G., Filmer D. Comparison of experimental binding data and theoretical models in proteins containing subunits. Biochemistry. 1966 Jan;5(1):365–385. doi: 10.1021/bi00865a047. [DOI] [PubMed] [Google Scholar]
  10. Kuo L. C., Zambidis I., Caron C. Triggering of allostery in an enzyme by a point mutation: ornithine transcarbamoylase. Science. 1989 Aug 4;245(4917):522–524. doi: 10.1126/science.2667139. [DOI] [PubMed] [Google Scholar]
  11. MONOD J., WYMAN J., CHANGEUX J. P. ON THE NATURE OF ALLOSTERIC TRANSITIONS: A PLAUSIBLE MODEL. J Mol Biol. 1965 May;12:88–118. doi: 10.1016/s0022-2836(65)80285-6. [DOI] [PubMed] [Google Scholar]
  12. Marcq S., Diaz-Ruano A., Charlier P., Dideberg O., Tricot C., Piérard A., Stalon V. Molecular size and symmetry of Pseudomonas aeruginosa catabolic ornithine carbamoyltransferase. An X-ray crystallography analysis. J Mol Biol. 1991 Jul 5;220(1):9–12. doi: 10.1016/0022-2836(91)90375-g. [DOI] [PubMed] [Google Scholar]
  13. Nguyen V. T., Baker D. P., Tricot C., Baur H., Villeret V., Dideberg O., Gigot D., Stalon V., Haas D. Catabolic ornithine carbamoyltransferase of Pseudomonas aeruginosa. Importance of the N-terminal region for dodecameric structure and homotropic carbamoylphosphate cooperativity. Eur J Biochem. 1996 Feb 15;236(1):283–293. doi: 10.1111/j.1432-1033.1996.00283.x. [DOI] [PubMed] [Google Scholar]
  14. Nguyen V. T., Tricot C., Stalon V., Dideberg O., Villeret V., Haas D. Methionine-321 in the C-terminal alpha-helix of catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa is important for positive homotropic cooperativity. FEMS Microbiol Lett. 1994 Dec 15;124(3):411–417. doi: 10.1111/j.1574-6968.1994.tb07317.x. [DOI] [PubMed] [Google Scholar]
  15. Prescott L. M., Jones M. E. Modified methods for the determination of carbamyl aspartate. Anal Biochem. 1969 Dec;32(3):408–419. doi: 10.1016/s0003-2697(69)80008-4. [DOI] [PubMed] [Google Scholar]
  16. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Sinning I., Kleywegt G. J., Cowan S. W., Reinemer P., Dirr H. W., Huber R., Gilliland G. L., Armstrong R. N., Ji X., Board P. G. Structure determination and refinement of human alpha class glutathione transferase A1-1, and a comparison with the Mu and Pi class enzymes. J Mol Biol. 1993 Jul 5;232(1):192–212. doi: 10.1006/jmbi.1993.1376. [DOI] [PubMed] [Google Scholar]
  18. Stalon V., Legrain C., Wiame J. M. Anabolic ornithine carbamolytransferase of Pseudomonas. The bases of its functional specialization. Eur J Biochem. 1977 Apr 1;74(2):319–327. doi: 10.1111/j.1432-1033.1977.tb11396.x. [DOI] [PubMed] [Google Scholar]
  19. Stalon V., Ramos F., Piérard A., Wiame J. M. Regulation of the catabolic ornithine carbamoyltransferase of Pseudomonas fluorescens. A comparison with the anabolic transferase and with a mutationally modified catabolic transferase. Eur J Biochem. 1972 Aug 18;29(1):25–35. doi: 10.1111/j.1432-1033.1972.tb01953.x. [DOI] [PubMed] [Google Scholar]
  20. Stebbins J. W., Kantrowitz E. R. Conversion of the noncooperative Bacillus subtilis aspartate transcarbamoylase into a cooperative enzyme by a single amino acid substitution. Biochemistry. 1992 Mar 3;31(8):2328–2332. doi: 10.1021/bi00123a017. [DOI] [PubMed] [Google Scholar]
  21. Tricot C., Nguyen V. T., Stalon V. Steady-state kinetics and analysis of pH dependence on wild-type and a modified allosteric Pseudomonas aeruginosa ornithine carbamoyltransferase containing the replacement of glutamate 105 by alanine. Eur J Biochem. 1993 Aug 1;215(3):833–839. doi: 10.1111/j.1432-1033.1993.tb18099.x. [DOI] [PubMed] [Google Scholar]
  22. Tricot C., Nguyen V. T., Stalon V. Steady-state kinetics and analysis of pH dependence on wild-type and a modified allosteric Pseudomonas aeruginosa ornithine carbamoyltransferase containing the replacement of glutamate 105 by alanine. Eur J Biochem. 1993 Aug 1;215(3):833–839. doi: 10.1111/j.1432-1033.1993.tb18099.x. [DOI] [PubMed] [Google Scholar]
  23. Vander Wauven C., Piérard A., Kley-Raymann M., Haas D. Pseudomonas aeruginosa mutants affected in anaerobic growth on arginine: evidence for a four-gene cluster encoding the arginine deiminase pathway. J Bacteriol. 1984 Dec;160(3):928–934. doi: 10.1128/jb.160.3.928-934.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Villeret V., Tricot C., Stalon V., Dideberg O. Crystal structure of Pseudomonas aeruginosa catabolic ornithine transcarbamoylase at 3.0-A resolution: a different oligomeric organization in the transcarbamoylase family. Proc Natl Acad Sci U S A. 1995 Nov 7;92(23):10762–10766. doi: 10.1073/pnas.92.23.10762. [DOI] [PMC free article] [PubMed] [Google Scholar]

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