Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1990 Feb;172(2):997–1004. doi: 10.1128/jb.172.2.997-1004.1990

Purification and characterization of fatty acid beta-oxidation enzymes from Caulobacter crescentus.

M A O'Connell 1, G Orr 1, L Shapiro 1
PMCID: PMC208528  PMID: 1967602

Abstract

Acetoacetyl coenzyme A (acetoacetyl-CoA) thiolase, an enzyme required for short-chain fatty acid degradation, has been purified to near homogeneity from Caulobacter crescentus. The relative heat stability of this enzyme allowed it to be separated from beta-ketoacyl-CoA thiolase. The purification scheme minus the heating step also permitted the copurification of crotonase and 3-hydroxyacyl-CoA dehydrogenase. These activities are in a multienzyme complex in Escherichia coli, but a similar complex was not observed in C. crescentus. Instead, separate proteins differing in enzymatic activity were detected, analogous to the beta-oxidation enzymes that have been isolated from Clostridium acetobutylicum and from mitochondria of higher eucaryotes. In these cells, as appears to be the case with C. crescentus, the individual enzymes form multimers of identical subunits.

Full text

PDF
997

Images in this article

1000FIG. 3
on p.1000

Selected References

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

  1. Binstock J. F., Pramanik A., Schulz H. Isolation of a multi-enzyme complex of fatty acid oxidation from Escherichia coli. Proc Natl Acad Sci U S A. 1977 Feb;74(2):492–495. doi: 10.1073/pnas.74.2.492. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Binstock J. F., Schulz H. Fatty acid oxidation complex from Escherichia coli. Methods Enzymol. 1981;71(Pt 100):403–411. doi: 10.1016/0076-6879(81)71051-6. [DOI] [PubMed] [Google Scholar]
  3. Contreras I., Bender R. A., Mansour J., Henry S., Shapiro L. Caulobacter cresentus mutant defective in membrane phospholipid synthesis. J Bacteriol. 1979 Nov;140(2):612–619. doi: 10.1128/jb.140.2.612-619.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Contreras I., Shapiro L., Henry S. Membrane phospholipid composition of Caulobacter crescentus. J Bacteriol. 1978 Sep;135(3):1130–1136. doi: 10.1128/jb.135.3.1130-1136.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Duncombe G. R., Frerman F. E. Molecular and catalytic properties of the acetoacetyl-coenzyme A thiolase of Escherichia coli. Arch Biochem Biophys. 1976 Sep;176(1):159–170. doi: 10.1016/0003-9861(76)90152-1. [DOI] [PubMed] [Google Scholar]
  6. Feigenbaum J., Schulz H. Thiolases of Escherichia coli: purification and chain length specificities. J Bacteriol. 1975 May;122(2):407–411. doi: 10.1128/jb.122.2.407-411.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fong J. C., Schulz H. Short-chain and long-chain enoyl-CoA hydratases from pig heart muscle. Methods Enzymol. 1981;71(Pt 100):390–398. doi: 10.1016/0076-6879(81)71049-8. [DOI] [PubMed] [Google Scholar]
  8. Haga T., Haga K., Gilman A. G. Hydrodynamic properties of the beta-adrenergic receptor and adenylate cyclase from wild type and varient S49 lymphoma cells. J Biol Chem. 1977 Aug 25;252(16):5776–5782. [PubMed] [Google Scholar]
  9. Hall C. L. Acyl-CoA dehydrogenases from pig liver mitochondria. Methods Enzymol. 1981;71(Pt 100):375–385. doi: 10.1016/0076-6879(81)71047-4. [DOI] [PubMed] [Google Scholar]
  10. Hodgson D., Shaw P., Letts V., Henry S., Shapiro L. Genetic analysis and characterization of a Caulobacter crescentus mutant defective in membrane biogenesis. J Bacteriol. 1984 May;158(2):430–440. doi: 10.1128/jb.158.2.430-440.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hodgson D., Shaw P., O'Connell M., Henry S., Shapiro L. Caulobacter crescentus fatty acid-dependent cell cycle mutant. J Bacteriol. 1984 Apr;158(1):156–162. doi: 10.1128/jb.158.1.156-162.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jenkins L. S., Nunn W. D. Genetic and molecular characterization of the genes involved in short-chain fatty acid degradation in Escherichia coli: the ato system. J Bacteriol. 1987 Jan;169(1):42–52. doi: 10.1128/jb.169.1.42-52.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  15. 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]
  16. Letts V., Shaw P., Shapiro L., Henry S. Synthesis and utilization of fatty acids by wild-type and fatty acid auxotrophs of Caulobacter crescentus. J Bacteriol. 1982 Sep;151(3):1269–1278. doi: 10.1128/jb.151.3.1269-1278.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. MARTIN R. G., AMES B. N. A method for determining the sedimentation behavior of enzymes: application to protein mixtures. J Biol Chem. 1961 May;236:1372–1379. [PubMed] [Google Scholar]
  18. Maloy S. R., Bohlander M., Nunn W. D. Elevated levels of glyoxylate shunt enzymes in Escherichia coli strains constitutive for fatty acid degradation. J Bacteriol. 1980 Aug;143(2):720–725. doi: 10.1128/jb.143.2.720-725.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Morrissey J. H. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem. 1981 Nov 1;117(2):307–310. doi: 10.1016/0003-2697(81)90783-1. [DOI] [PubMed] [Google Scholar]
  20. Nunn W. D. A molecular view of fatty acid catabolism in Escherichia coli. Microbiol Rev. 1986 Jun;50(2):179–192. doi: 10.1128/mr.50.2.179-192.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. O'Brien W. J., Frerman F. E. Evidence for a complex of three beta-oxidation enzymes in Escherichia coli: induction and localization. J Bacteriol. 1977 Nov;132(2):532–540. doi: 10.1128/jb.132.2.532-540.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. O'Connell M., Henry S., Shapiro L. Fatty acid degradation in Caulobacter crescentus. J Bacteriol. 1986 Oct;168(1):49–54. doi: 10.1128/jb.168.1.49-54.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pauli G., Ehring R., Overath P. Fatty acid degradation in Escherichia coli: requirement of cyclic adenosine monophosphate and cyclic adenosine monophosphate receptor protein for enzyme synthesis. J Bacteriol. 1974 Mar;117(3):1178–1183. doi: 10.1128/jb.117.3.1178-1183.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pauli G., Overath P. ato Operon: a highly inducible system for acetoacetate and butyrate degradation in Escherichia coli. Eur J Biochem. 1972 Sep 25;29(3):553–562. doi: 10.1111/j.1432-1033.1972.tb02021.x. [DOI] [PubMed] [Google Scholar]
  25. Pawar S., Schulz H. The structure of the multienzyme complex of fatty acid oxidation from Escherichia coli. J Biol Chem. 1981 Apr 25;256(8):3894–3899. [PubMed] [Google Scholar]
  26. Pramanik A., Pawar S., Antonian E., Schulz H. Five different enzymatic activities are associated with the multienzyme complex of fatty acid oxidation from Escherichia coli. J Bacteriol. 1979 Jan;137(1):469–473. doi: 10.1128/jb.137.1.469-473.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Shapiro L., Gober J. W. Positioning of gene products during Caulobacter cell differentiation. J Cell Sci Suppl. 1989;11:85–97. doi: 10.1242/jcs.1989.supplement_11.7. [DOI] [PubMed] [Google Scholar]
  28. Spratt S. K., Black P. N., Ragozzino M. M., Nunn W. D. Cloning, mapping, and expression of genes involved in the fatty acid-degradative multienzyme complex of Escherichia coli. J Bacteriol. 1984 May;158(2):535–542. doi: 10.1128/jb.158.2.535-542.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sramek S. J., Frerman F. E. Purification and properties of Escherichia coli coenzyme A-transferase. Arch Biochem Biophys. 1975 Nov;171(1):14–26. doi: 10.1016/0003-9861(75)90002-8. [DOI] [PubMed] [Google Scholar]
  30. Thorpe C. Acyl-CoA dehydrogenase from pig kidney. Methods Enzymol. 1981;71(Pt 100):366–374. doi: 10.1016/0076-6879(81)71046-2. [DOI] [PubMed] [Google Scholar]
  31. Waterson R. M., Castellino F. J., Hass G. M., Hill R. L. Purification and characterization of crotonase from Clostridium acetobutylicum. J Biol Chem. 1972 Aug 25;247(16):5266–5271. [PubMed] [Google Scholar]
  32. Weeks G., Shapiro M., Burns R. O., Wakil S. J. Control of fatty acid metabolism. I. Induction of the enzymes of fatty acid oxidation in Escherichia coli. J Bacteriol. 1969 Feb;97(2):827–836. doi: 10.1128/jb.97.2.827-836.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES