1887

Microbiology Society logo

Volume 141, Issue 10

Acetyl-CoA-dependent pyruvate carboxylase from the photosynthetic bacterium : rapid and efficient purification using dye-ligand affinity chromatography Free

Abstract

Summary: Pyruvate carboxylase (PC) was purified to homogeneity from an overexpressing strain of the purple photosynthetic bacterium using a rapid dye-ligand affinity chromatography procedure, in which dye-bound enzyme was specifically eluted with a low concentration of acetyl-CoA, an allosteric activator of the enzyme. The enzyme purified by this method was obtained in 75% yield with a specific activity of 40 U (mg protein). In contrast, affinity chromatography on a monomeric avidin column, commonly used in the purification of biotin-containing carboxylases, resulted in a yield of < 40%, with a specific activity of 10 U (mg protein) The enzyme purified by the dye-linked procedure had a subunit molecular mass of 140000 Da and was absolutely dependent on acetyl-CoA for activity. Acetyl-CoA was also effective in protecting the enzyme from thermal denaturation. The enzyme was inhibited by 2-oxoglutarate and, to a lesser extent, L-aspartate, with sigmoidal kinetics with respect to acetyl-CoA concentration. The amino acid composition, pH optimum and kinetic constants for pyruvate, ATP and bicarbonate were determined. An N-terminal sequence of 26 residues was obtained, which was homologous to the N-terminal regions of several eukaryotic PCs, propionyl-CoA carboxylases and acetyl-CoA carboxylase.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): acetyl-CoA , dye-ligand chromatography , Keywords: pyruvate carboxvylase , N-terminal sequence and Rhodobacter capsulatus
© Society for General Microbiology 1995
Loading

Article metrics loading...

/content/journal/micro/10.1099/13500872-141-10-2619
1995-10-01
2024-04-26
Loading full text...

Full text loading...

/deliver/fulltext/micro/141/10/mic-141-10-2619.html?itemId=/content/journal/micro/10.1099/13500872-141-10-2619&mimeType=html&fmt=ahah

References

  1. Ashman L.K., Keech D.B., Wallace J.C., Nielsen J. 1972; Sheep kidney pyruvate carboxylase.. J Biol Chem 247:5818–5824
     [Google Scholar]
  2. Barden R.E., Taylor B.L., Ishohashi F., Frey W.H., Zander G., Lee J.C., Utter M.F. 1975; Structural properties of pyruvate carboxylases from chicken liver and other sources.. Proc Nail Acad Sci 72:4308–4312
     [Google Scholar]
  3. Buckley J.J., Libor S., Sundaram T.K. 1969; Biotin subunits of acetyl CoA carboxylase and pyruvate carboxylase from a thermophilic Bacillus.. Arch Biochem Biophjs 192:396–402
     [Google Scholar]
  4. Cazzulo J.i., Stoppani A.O.M. 1968; The regulation of yeast pyruvate carboxylase by acetyl-CoA and L-aspartate.. Arch Biochem Biophjs 127:563–570
     [Google Scholar]
  5. Cazzulo J.J., Stoppani A.O.M. 1969a; Effects of magnesium, manganese and adenosine triphosphate ions on pyruvate carboxylase from bakers yeast.. Biochem J 112:747–754
     [Google Scholar]
  6. Cazzulo J.J., Stoppani A.O.M. 1969b; Effects of adenosine phosphates and nicotinamide nucleotides on pyruvate carboxylase from bakers yeast.. Biochem J 112:755–762
     [Google Scholar]
  7. Cazzulo J.J., Sundaram T.K., Kornberg H.L. 1970; Properties and regulation of pyruvate carboxylase from Bacillus stearo- thermophilus.. Proc R Soc Lond B176:1–19
     [Google Scholar]
  8. Colbeau A., Godfrey A., Vignais P.M. 1986; Cloning of DNA fragments carrying hydrogenase genes of Rhodobacter capsulatus.. Biochemie 68:147–155
     [Google Scholar]
  9. Devereux J., Haeberli P., Smithies O. 1984; A comprehensive set of sequence analysis programs for the VAX.. Nucleic Acids Res 12:387–395
     [Google Scholar]
  10. Dixon G.H., Kornberg H.L. 1959; Assay methods for key enzymes of the glyoxylate cycle.. Biochem J 72:3P
     [Google Scholar]
  11. Easterbrook-Smith S.B., Hudson P.J., Goss N.H., Keech D.B., Wallace J.C. 1976; Pyruvate carboxylase: mechanism of the second partial reaction.. Arch Biochem Biophjs 176:709–716
     [Google Scholar]
  12. Feir H.A., Suzuki I. 1969; Pyruvate carboxylase of Aspergillus niger: kinetic study of a biotin containing carboxylase.. Can J Biochem 47:697–710
     [Google Scholar]
  13. Fox D.K., Roseman S. 1986; Isolation and characterisation of homogeneous acetate kinase from Escherichia coli and Salmonella tjphimurium.. J Biol Chem 261:3487–3497
     [Google Scholar]
  14. Guest J.R., Creaghan I.T. 1973; Gene-protein relationships of the α-keto acid dehydrogenase complexes of Escherichia coli K12: isolation and characterization of lipoamide dehydrogenase mutants.. J Gen Microbiol 75:197–210
     [Google Scholar]
  15. Hamblin M.J., Shaw J.G., Curson J.P., Kelly D.J. 1990; Mutagenesis, cloning and complementation analysis of C4-dicarb- oxylate transport genes from Rhodobacter capsulatus.. Mol Microbiol 4:1567–1574
     [Google Scholar]
  16. Hanahan D. 1985; Techniques for transformation of E. coli. . In DNA Cloning, a Practical Approach 1 pp. 109–135 Glover D. Edited by Oxford: IRL Press;
     [Google Scholar]
  17. Hartman R.E., Keen N.T. 1974; The pyruvate carboxylase of Verticillium abo-atrum.. J Gen Microbiol 81:15–19
     [Google Scholar]
  18. Henrikson K.P., Allen H.G., Malloy W.L. 1979; An avidin monomer affinity column for the purification of biotin containing enzymes.. Anal Biocbem 94:366–370
     [Google Scholar]
  19. Hess B., Wieker H.J. 1974; Pyruvate kinase from yeast.. In Methods of Enzymatic Analysis 2 pp. 778–783 Bergmeyer H.U. Edited by New York: Academic Press;
     [Google Scholar]
  20. Hillmer P., Gest H. 1977; H2 metabolism in the photosynthetic bacterium Rhodobacter capsulatus: H2 production by growing cultures.. J Bacteriol 129:724–731
     [Google Scholar]
  21. Keech D.B., Barrit G.J. 1967; Allosteric activation of sheep kidney pyruvate carboxylase by the magnesium ion (Mg2+) and the magnesium adenosine triphosphate ion (MgATP2-).. J Biol Chem 242:1983–1987
     [Google Scholar]
  22. Keech D.B., Wallace J.C. 1985 Pjruvate carboxjlase.CRC Series in Enzyme Biology. Boca Raton, Florida: CRC Press;
     [Google Scholar]
  23. Keen N.T., Tamaki S., Kobayashi D., Trollinger D. 1988; Improved broad host range plasmids for DNA cloning in Gramnegative bacteria.. Gene 70:191–197
     [Google Scholar]
  24. Leyland M.L., Kelly D.J. 1991; Purification and charac-terisation of a monomeric isocitrate dehydrogenase with dual coenzyme specificity from the photosynthetic bacterium Rhodo- microbium vannielii.. Eur J Biochem 202:85–93
     [Google Scholar]
  25. Lim F., Morris C.P., Occhiodoro F., Wallace J.C. 1988; Sequence and domain structure of yeast pyruvate carboxylase.. J Biol Chem 263:11493–11497
     [Google Scholar]
  26. Marchelonis J.J., Weltman J.K. 1971; Relatedness among proteins: a new method estimation and its application to immuno-globulins.. Comp Biochem Physiol 38B:609–625
     [Google Scholar]
  27. Milard de Forchetti S.R., Cazzulo J.J. 1976; Some properties of the pyruvate carboxylase from Pseudomonas fluorescens. . J Gen Microbiol 93:75–81
     [Google Scholar]
  28. Payne J., Morris J.G. 1969; Pyruvate carboxylase in Rhodo- pseudomonas sphaeroides.. J Gen Microbiol 59:97–101
     [Google Scholar]
  29. Phillips N.F.B., Snoswell M.A., Chapman-Smith A., Keech B., Wallace J.C. 1992; Isolation of a carboxyphosphate intermediate and the locus of acetyl-CoA action in the pyruvate carboxylase reaction.. Biochemistry 31:9445–9450
     [Google Scholar]
  30. Ruiz-Amil M., de Torrontegui E., Catalina L., Losada M. 1965; Properties and function of yeast pyruvate carboxylase.. J Biol Chem 240:3485–3492
     [Google Scholar]
  31. Sambrook J., Fritsch E.F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual, 2nd edn.. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  32. Schobert P., Bowien B. 1984; Unusual C3 and C4 metabolism in the chemoautotroph Alcaligenes eutrophus.. JBacteriol 159:161–172
     [Google Scholar]
  33. Scopes R.K. 1986; Strategies for enzyme isolation using dye- ligand and related adsorbents.. J Chromatogr 376:131–140
     [Google Scholar]
  34. Scrutton M.C., Taylor B.L. 1974; Isolation and characterisation of pyruvate carboxylase from Azotobacter vinelandii.. Arch Biochem Biophys 164:641–684
     [Google Scholar]
  35. Scrutton M.C., Utter M.F. 1965; Pyruvate carboxylase III. Some physical and chemical properties of the highly purified enzyme.. J Biol Chem 240:1–9
     [Google Scholar]
  36. Scrutton M.C., Young M.R. 1972; Pyruvate carboxylase.. In The Enzymes, 3rd edn. 6 pp. 1–35 Boyer P.D. Edited by New York: Academic Press;
     [Google Scholar]
  37. Scrutton M.C., Olmsted M.R., Utter M.F. 1969; Pyruvate carboxylase from chicken liver.. Methods Enzymol 13:235–248
     [Google Scholar]
  38. Simon R., Priefer V. 1983; A broad host range mobilisation system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria.. Bio/technology 1:784–791
     [Google Scholar]
  39. Stan H.J., Schormuller J. 1968; Regulation of pyruvate carboxylase of Penicillium camemberti by the adenylate system.. Biochem Biophys Res Commun 32:289–294
     [Google Scholar]
  40. Suebert W., Weicker H. 1969; Pyruvate carboxylase from Pseudomonas.. Methods Envymol 13:258–262
     [Google Scholar]
  41. Taylor B.L., Barden R.E., Scrutton M.C. 1972; Identification of the reacting form of pyruvate carboxylase.. J Biol Chem 247:7383–7390
     [Google Scholar]
  42. Taylor D.P., Cohen S.N., Clark W.G., Marrs B.L. 1983; Alignment of the genetic and restriction maps of the photosynthesis regions of the Rhodopseudomonas capsulata chromosome by a conjugation-mediated marker rescue technique.. J Bacteriol 154:580–590
     [Google Scholar]
  43. Utter M.F., Kolenbrander H.M. 1972; Formation of oxalo- acetate by CO2 fixation on phosphoenolpyruvate.. In The Enzymes, 3rd edn. 6 pp. 117–168 Boyer P.D. Edited by New York: Academic Press;
     [Google Scholar]
  44. Wallace J.C., Easterbrook-Smith S.B. 1985; The structure of pyruvate carboxylase.. In Pyruvate carboxylase pp. 65–108 Keech B.D., Wallace J.C. Edited by Boca Raton, Florida: CRC Press;
     [Google Scholar]
  45. Warren G.B., Tipton K.F. 1974a; Pig liver pyruvate carb-oxylase. Purification, properties and cation specificity.. Biochem J 139:299–310
     [Google Scholar]
  46. Warren G.B., Tipton K.F. 1974b; Pig liver pyruvate carb-oxylase. The reaction pathway for the carboxylation of pyruvate.. Biochem J 139:311–320
     [Google Scholar]
  47. Weaver P.F., Wall J.D., Gest H. 1975; Characterisation of Rhodopseudomonas capsulata.. Arch Microbiol 105:207–216
     [Google Scholar]
  48. Willison J.C. 1988; Pyruvate and acetate metabolism in the photosynthetic bacterium Rhodobacter capsulatus.. J Gen Microbiol 34:2429–2439
     [Google Scholar]
  49. Wooford N.Q., Beaty R.S., Mdnerney M.J. 1986; Prepar-ation of cell-free extracts and the enzymes involved in fatty-acid metabolism of Syntrophomonas wolfei.. J Bacteriol 167:179–185
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/13500872-141-10-2619
Loading
Acetyl-CoA-dependent pyruvate carboxylase from the photosynthetic bacterium Rhodobacter capsulatus: rapid and efficient purification using dye-ligand affinity chromatography
Microbiology 141, 2619 (1995); https://doi.org/10.1099/13500872-141-10-2619
/content/journal/micro/10.1099/13500872-141-10-2619
/content/journal/micro/10.1099/13500872-141-10-2619
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error