Skip to main content
NCBI home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1993 Apr;59(4):1149–1154. doi: 10.1128/aem.59.4.1149-1154.1993

Purification of Pseudomonas putida acyl coenzyme A ligase active with a range of aliphatic and aromatic substrates.

M Fernández-Valverde 1, A Reglero 1, H Martinez-Blanco 1, J M Luengo 1
PMCID: PMC202253  PMID: 8476289

Abstract

Acyl coenzyme A (acyl-CoA) ligase (acyl-CoA synthetase [ACoAS]) from Pseudomonas putida U was purified to homogeneity (252-fold) after this bacterium was grown in a chemically defined medium containing octanoic acid as the sole carbon source. The enzyme, which has a mass of 67 kDa, showed maximal activity at 40 degrees C in 10 mM K2PO4H-NaPO4H2 buffer (pH 7.0) containing 20% (wt/vol) glycerol. Under these conditions, ACoAS showed hyperbolic behavior against acetate, CoA, and ATP; the Kms calculated for these substrates were 4.0, 0.7, and 5.2 mM, respectively. Acyl-CoA ligase recognizes several aliphatic molecules (acetic, propionic, butyric, valeric, hexanoic, heptanoic, and octanoic acids) as substrates, as well as some aromatic compounds (phenylacetic and phenoxyacetic acids). The broad substrate specificity of ACoAS from P. putida was confirmed by coupling it with acyl-CoA:6-aminopenicillanic acid acyltransferase from Penicillium chrysogenum to study the formation of several penicillins.

Full text

PDF
1149

Images in this article

1152Image
on p.1152

Selected References

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

  1. Alonso M. J., Bermejo F., Reglero A., Fernández-Cañn J. M., González de Buitrago G., Luengo J. M. Enzymatic synthesis of penicillins. J Antibiot (Tokyo) 1988 Aug;41(8):1074–1084. doi: 10.7164/antibiotics.41.1074. [DOI] [PubMed] [Google Scholar]
  2. Altenschmidt U., Oswald B., Fuchs G. Purification and characterization of benzoate-coenzyme A ligase and 2-aminobenzoate-coenzyme A ligases from a denitrifying Pseudomonas sp. J Bacteriol. 1991 Sep;173(17):5494–5501. doi: 10.1128/jb.173.17.5494-5501.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Anson J. G., Mackinnon G. Novel pseudomonas plasmid involved in aniline degradation. Appl Environ Microbiol. 1984 Oct;48(4):868–869. doi: 10.1128/aem.48.4.868-869.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barnsley E. A. The induction of the enzymes of naphthalene metabolism in pseudomonads by salicylate and 2-aminobenzoate. J Gen Microbiol. 1975 May;88(1):193–196. doi: 10.1099/00221287-88-1-193. [DOI] [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  6. Brunner R., Rohr M. Phenacyl:coenzyme A ligase. Methods Enzymol. 1975;43:476–481. doi: 10.1016/0076-6879(75)43107-x. [DOI] [PubMed] [Google Scholar]
  7. Byng G. S., Johnson J. L., Whitaker R. J., Gherna R. L., Jensen R. A. The evolutionary pattern of aromatic amino acid biosynthesis and the emerging phylogeny of pseudomonad bacteria. J Mol Evol. 1983;19(3-4):272–282. doi: 10.1007/BF02099974. [DOI] [PubMed] [Google Scholar]
  8. Chaudhry G. R., Chapalamadugu S. Biodegradation of halogenated organic compounds. Microbiol Rev. 1991 Mar;55(1):59–79. doi: 10.1128/mr.55.1.59-79.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Connerton I. F., Fincham J. R., Sandeman R. A., Hynes M. J. Comparison and cross-species expression of the acetyl-CoA synthetase genes of the Ascomycete fungi, Aspergillus nidulans and Neurospora crassa. Mol Microbiol. 1990 Mar;4(3):451–460. doi: 10.1111/j.1365-2958.1990.tb00611.x. [DOI] [PubMed] [Google Scholar]
  10. Dagley S. Catabolism of aromatic compounds by micro-organisms. Adv Microb Physiol. 1971;6(0):1–46. doi: 10.1016/s0065-2911(08)60066-1. [DOI] [PubMed] [Google Scholar]
  11. Evans W. C. Biochemistry of the bacterial catabolism of aromatic compounds in anaerobic environments. Nature. 1977 Nov 3;270(5632):17–22. doi: 10.1038/270017a0. [DOI] [PubMed] [Google Scholar]
  12. Evans W. C., Fuchs G. Anaerobic degradation of aromatic compounds. Annu Rev Microbiol. 1988;42:289–317. doi: 10.1146/annurev.mi.42.100188.001445. [DOI] [PubMed] [Google Scholar]
  13. Frenkel E. P., Kitchens R. L. Purification and properties of acetyl coenzyme A synthetase from bakers' yeast. J Biol Chem. 1977 Jan 25;252(2):504–507. [PubMed] [Google Scholar]
  14. Geissler J. F., Harwood C. S., Gibson J. Purification and properties of benzoate-coenzyme A ligase, a Rhodopseudomonas palustris enzyme involved in the anaerobic degradation of benzoate. J Bacteriol. 1988 Apr;170(4):1709–1714. doi: 10.1128/jb.170.4.1709-1714.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gibson D. T. Microbial degradation of aromatic compounds. Science. 1967 Sep 13;161(3846):1093–1097. [PubMed] [Google Scholar]
  16. Horn J. M., Harayama S., Timmis K. N. DNA sequence determination of the TOL plasmid (pWWO) xylGFJ genes of Pseudomonas putida: implications for the evolution of aromatic catabolism. Mol Microbiol. 1991 Oct;5(10):2459–2474. doi: 10.1111/j.1365-2958.1991.tb02091.x. [DOI] [PubMed] [Google Scholar]
  17. Hosaka K., Mishina M., Tanaka T., Kamiryo T., Numa S. Acyl-coenzyme-A synthetase I from Candida lipolytica. Purification, properties and immunochemical studies. Eur J Biochem. 1979 Jan 2;93(1):197–203. doi: 10.1111/j.1432-1033.1979.tb12811.x. [DOI] [PubMed] [Google Scholar]
  18. Imesch E., Rous S. Partial purification of rat liver cytoplasmic acetyl-CoA synthetase; characterization of some properties. Int J Biochem. 1984;16(8):875–881. doi: 10.1016/0020-711x(84)90146-0. [DOI] [PubMed] [Google Scholar]
  19. Jetten M. S., Stams A. J., Zehnder A. J. Isolation and characterization of acetyl-coenzyme A synthetase from Methanothrix soehngenii. J Bacteriol. 1989 Oct;171(10):5430–5435. doi: 10.1128/jb.171.10.5430-5435.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kameda K., Nunn W. D. Purification and characterization of acyl coenzyme A synthetase from Escherichia coli. J Biol Chem. 1981 Jun 10;256(11):5702–5707. [PubMed] [Google Scholar]
  21. Koenig K., Andreesen J. R. Molybdenum Involvement in Aerobic Degradation of 2-Furoic Acid by Pseudomonas putida Fu1. Appl Environ Microbiol. 1989 Jul;55(7):1829–1834. doi: 10.1128/aem.55.7.1829-1834.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  23. Martinez-Blanco H., Reglero A., Luengo J. M. Carbon catabolite regulation of phenylacetyl-CoA ligase from Pseudomonas putida. Biochem Biophys Res Commun. 1990 Mar 30;167(3):891–897. doi: 10.1016/0006-291x(90)90607-o. [DOI] [PubMed] [Google Scholar]
  24. Martínez-Blanco H., Reglero A., Fernández-Valverde M., Ferrero M. A., Moreno M. A., Peñalva M. A., Luengo J. M. Isolation and characterization of the acetyl-CoA synthetase from Penicillium chrysogenum. Involvement of this enzyme in the biosynthesis of penicillins. J Biol Chem. 1992 Mar 15;267(8):5474–5481. [PubMed] [Google Scholar]
  25. Martínez-Blanco H., Reglero A., Luengo J. M. "In vitro" synthesis of different naturally-occurring, semisynthetic and synthetic penicillins using a new and effective enzymatic coupled system. J Antibiot (Tokyo) 1991 Nov;44(11):1252–1258. doi: 10.7164/antibiotics.44.1252. [DOI] [PubMed] [Google Scholar]
  26. Martínez-Blanco H., Reglero A., Martín-Villacorta J., Luengo J. M. Design of an enzymatic hybrid system: a useful strategy for the biosynthesis of benzylpenicillin in vitro. FEMS Microbiol Lett. 1990 Oct;60(1-2):113–116. doi: 10.1111/j.1574-6968.1990.tb03872.x. [DOI] [PubMed] [Google Scholar]
  27. Martínez-Blanco H., Reglero A., Rodriguez-Aparicio L. B., Luengo J. M. Purification and biochemical characterization of phenylacetyl-CoA ligase from Pseudomonas putida. A specific enzyme for the catabolism of phenylacetic acid. J Biol Chem. 1990 Apr 25;265(12):7084–7090. [PubMed] [Google Scholar]
  28. Ramos J. L., Timmis K. N. Experimental evolution of catabolic pathways of bacteria. Microbiol Sci. 1987 Aug;4(8):228–237. [PubMed] [Google Scholar]
  29. Rodríguez-Aparicio L. B., Reglero A., Martínez-Blanco H., Luengo J. M. Fluorometric determination of phenylacetyl-CoA ligase from Pseudomonas putida: a very sensitive assay for a newly described enzyme. Biochim Biophys Acta. 1991 Mar 4;1073(2):431–433. doi: 10.1016/0304-4165(91)90153-8. [DOI] [PubMed] [Google Scholar]
  30. Wheelis L. The genetics of dissimilarity pathways in Pseudomonas. Annu Rev Microbiol. 1975;29:505–524. doi: 10.1146/annurev.mi.29.100175.002445. [DOI] [PubMed] [Google Scholar]
  31. Wheelis M. L., Stanier R. Y. The genetic control of dissimilatory pathways in Pseudomonas putida. Genetics. 1970 Oct;66(2):245–266. doi: 10.1093/genetics/66.2.245. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES