Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 Aug;179(15):4859–4867. doi: 10.1128/jb.179.15.4859-4867.1997

The reductive tricarboxylic acid cycle of carbon dioxide assimilation: initial studies and purification of ATP-citrate lyase from the green sulfur bacterium Chlorobium tepidum.

T M Wahlund 1, F R Tabita 1
PMCID: PMC179334  PMID: 9244275

Abstract

Carbon dioxide is fixed largely by the reductive tricarboxylic acid (RTCA) cycle in green sulfur bacteria. One of the key enzymes, ATP-citrate lyase, was purified to apparent homogeneity from the moderately thermophilic green sulfur bacterium Chlorobium tepidum. The molecular weight of the native enzyme was about 550,000, and the preponderance of evidence indicated that the protein is composed of identical subunits (Mr of approximately 135,000) which degraded to two major proteins with Mrs of approximately 65,000 and approximately 42,000. Western immunoblots and in vitro phosphorylation experiments indicated that these two species could have been the result of proteolysis by an endogenous protease, similar to what has been observed with mammalian, yeast, and mold ATP-citrate lyase. In addition to apparent structural similarities, the catalytic properties of C. tepidum ATP-citrate lyase showed marked similarities to the eukaryotic enzyme, with significant differences from other prokaryotic ATP-citrate lyases, including the enzyme from the closely related organism Chlorobium limicola. Phosphorylation of C. tepidum ATP-citrate lyase occurred, presumably on a histidine residue at the active site, similar to the case for the mammalian enzyme. In contrast to the situation observed for other prokaryotic ATP-citrate lyase enzymes, the C. tepidum enzyme was not able to replace ATP and GTP for activity or use Cu2+ to replace Mg2+ for enzyme activity. Given the apparent structural and catalytic similarities of the enzyme from C. tepidum and its eukaryotic counterpart, the C. tepidum system should serve as an excellent model for studies of the enzymology and regulation of this protein.

Full Text

The Full Text of this article is available as a PDF (731.8 KB).

Selected References

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

  1. Alexander M. C., Kowaloff E. M., Witters L. A., Dennihy D. T., Avruch J. Purification of a hepatic 123,000-dalton hormone-stimulated 32P-peptide and its identification as ATP-citrate lyase. J Biol Chem. 1979 Aug 25;254(16):8052–8056. [PubMed] [Google Scholar]
  2. Antranikian G., Herzberg C., Gottschalk G. Characterization of ATP citrate lyase from Chlorobium limicola. J Bacteriol. 1982 Dec;152(3):1284–1287. doi: 10.1128/jb.152.3.1284-1287.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chen C., Gibbs M. Some Enzymes and Properties of the Reductive Carboxylic Acid Cycle Are Present in the Green Alga Chlamydomonas reinhardtii F-60. Plant Physiol. 1992 Feb;98(2):535–539. doi: 10.1104/pp.98.2.535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Elshourbagy N. A., Near J. C., Kmetz P. J., Sathe G. M., Southan C., Strickler J. E., Gross M., Young J. F., Wells T. N., Groot P. H. Rat ATP citrate-lyase. Molecular cloning and sequence analysis of a full-length cDNA and mRNA abundance as a function of diet, organ, and age. J Biol Chem. 1990 Jan 25;265(3):1430–1435. [PubMed] [Google Scholar]
  5. Evans M. C., Buchanan B. B., Arnon D. I. A new ferredoxin-dependent carbon reduction cycle in a photosynthetic bacterium. Proc Natl Acad Sci U S A. 1966 Apr;55(4):928–934. doi: 10.1073/pnas.55.4.928. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fritsch H., Beevers H. ATP Citrate Lyase from Germinating Castor Bean Endosperm: Localization and some Properties. Plant Physiol. 1979 Apr;63(4):687–691. doi: 10.1104/pp.63.4.687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Guerritore A., Hanozet G. M. Presence and adaptive changes of citrate cleavage enzyme in the yeast Rhodotorula gracilis. Experientia. 1970 Jan 15;26(1):28–30. doi: 10.1007/BF01900371. [DOI] [PubMed] [Google Scholar]
  8. Guy P. S., Cohen P., Hardie D. G. Purification and physicochemical properties of ATP citrate (pro-3S) lyase from lactating rat mammary gland and studies of its reversible phosphorylation. Eur J Biochem. 1981 Feb;114(2):399–405. doi: 10.1111/j.1432-1033.1981.tb05160.x. [DOI] [PubMed] [Google Scholar]
  9. Ishii M., Igarashi Y., Kodama T. Purification and characterization of ATP:citrate lyase from Hydrogenobacter thermophilus TK-6. J Bacteriol. 1989 Apr;171(4):1788–1792. doi: 10.1128/jb.171.4.1788-1792.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Linn T. C., Srere P. A. Identification of ATP citrate lyase as a phosphoprotein. J Biol Chem. 1979 Mar 10;254(5):1691–1698. [PubMed] [Google Scholar]
  13. Måhlén A. Purification and some properties of ATP citrate lyase from Penicillium spiculisporum. Eur J Biochem. 1973 Jul 16;36(2):342–346. doi: 10.1111/j.1432-1033.1973.tb02918.x. [DOI] [PubMed] [Google Scholar]
  14. Pentyala S. N., Benjamin W. B. Effect of oxaloacetate and phosphorylation on ATP-citrate lyase activity. Biochemistry. 1995 Sep 5;34(35):10961–10969. doi: 10.1021/bi00035a001. [DOI] [PubMed] [Google Scholar]
  15. Plowman D. M., Cleland W. W. Purification and kinetic studies of the citrate cleavage enzyme. J Biol Chem. 1967 Sep 25;242(18):4239–4247. [PubMed] [Google Scholar]
  16. Pulich W. M., Baalen C. Purification and characterization of glucose dehydrogenase from a heterotrophically grown blue-green alga. Plant Physiol. 1976 Sep;58(3):393–397. doi: 10.1104/pp.58.3.393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Redshaw J. C., Loten E. G. A rapid purification of hepatic ATP citrate lyase using blue Sepharose. FEBS Lett. 1981 Jan 26;123(2):261–264. doi: 10.1016/0014-5793(81)80303-1. [DOI] [PubMed] [Google Scholar]
  18. SRERE P. A. The citrate cleavage enzyme. I. Distribution and purification. J Biol Chem. 1959 Oct;234:2544–2547. [PubMed] [Google Scholar]
  19. Shashi K., Bachhawat A. K., Joseph R. ATP:citrate lyase of Rhodotorula gracilis: purification and properties. Biochim Biophys Acta. 1990 Jan 29;1033(1):23–30. doi: 10.1016/0304-4165(90)90189-4. [DOI] [PubMed] [Google Scholar]
  20. Singh M., Richards E. G., Mukherjee A., Srere P. A. Structure of ATP citrate lyase from rat liver. Physicochemical studies and proteolytic modification. J Biol Chem. 1976 Sep 10;251(17):5242–5250. [PubMed] [Google Scholar]
  21. Sjoberg E. R., Varki A. Kinetic and spatial interrelationships between ganglioside glycosyltransferases and O-acetyltransferase(s) in human melanoma cells. J Biol Chem. 1993 May 15;268(14):10185–10196. [PubMed] [Google Scholar]
  22. Tuhácková Z., Krivánek J. GTP, a nonsubstrate of ATP citrate lyase, is a phosphodonor for the enzyme histidine autophosphorylation. Biochem Biophys Res Commun. 1996 Jan 5;218(1):61–66. doi: 10.1006/bbrc.1996.0012. [DOI] [PubMed] [Google Scholar]
  23. Wagner P. D., Vu N. D. Phosphorylation of ATP-citrate lyase by nucleoside diphosphate kinase. J Biol Chem. 1995 Sep 15;270(37):21758–21764. doi: 10.1074/jbc.270.37.21758. [DOI] [PubMed] [Google Scholar]
  24. Wahlund T. M., Madigan M. T. Genetic transfer by conjugation in the thermophilic green sulfur bacterium Chlorobium tepidum. J Bacteriol. 1995 May;177(9):2583–2588. doi: 10.1128/jb.177.9.2583-2588.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Weber K., Pringle J. R., Osborn M. Measurement of molecular weights by electrophoresis on SDS-acrylamide gel. Methods Enzymol. 1972;26:3–27. doi: 10.1016/s0076-6879(72)26003-7. [DOI] [PubMed] [Google Scholar]
  26. Wei Y. F., Matthews H. R. Identification of phosphohistidine in proteins and purification of protein-histidine kinases. Methods Enzymol. 1991;200:388–414. doi: 10.1016/0076-6879(91)00156-q. [DOI] [PubMed] [Google Scholar]
  27. Weiss V., Magasanik B. Phosphorylation of nitrogen regulator I (NRI) of Escherichia coli. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8919–8923. doi: 10.1073/pnas.85.23.8919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wells T. N. ATP-citrate lyase from rat liver. Characterisation of the citryl-enzyme complexes. Eur J Biochem. 1991 Jul 1;199(1):163–168. doi: 10.1111/j.1432-1033.1991.tb16105.x. [DOI] [PubMed] [Google Scholar]
  29. Wells T. N., Saxty B. A. Redox control of catalysis in ATP-citrate lysate from rat liver. Eur J Biochem. 1992 Feb 15;204(1):249–255. doi: 10.1111/j.1432-1033.1992.tb16631.x. [DOI] [PubMed] [Google Scholar]
  30. Wolodko W. T., Brownie E. R., O'Connor M. D., Bridger W. A. Thiophosphorylation as a probe for subunit interactions in Escherichia coli succinyl coenzyme A synthetase. Further evidence for catalytic cooperativity and substrate synergism. J Biol Chem. 1983 Dec 10;258(23):14116–14119. [PubMed] [Google Scholar]

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

RESOURCES