Skip to main content
NCBI home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1990 Mar;56(3):607–613. doi: 10.1128/aem.56.3.607-613.1990

Purification and properties of an acetoacetyl coenzyme A-reacting phosphotransbutyrylase from Clostridium beijerinckii ("Clostridium butylicum") NRRL B593.

D K Thompson 1, J S Chen 1
PMCID: PMC183394  PMID: 2317039

Abstract

During the study of acetoacetyl coenzyme A (CoA)-reacting enzymes of Clostridium beijerinckii NRRL B593, a phosphate-dependent acetoacetyl-CoA-utilizing activity was detected in protein fractions devoid of thiolase and phosphotransacetylase. Further purification of this acetoacetyl-CoA-utilizing activity yielded an enzyme which may be designated as phosphotransbutyrylase (PTB; phosphate butyryltransferase [EC 2.3.1.19]). PTB from C. beijerinckii NRRL B593 was purified 160-fold with a yield of 14% and, with the best fractions, purified 190-fold to near homogeneity. It showed a native Mr of 205,000 and a subunit Mr of 33,000. PTB activity was sensitive to pH changes within the physiological range of 6 to 8. PTB exhibited a broad substrate specificity. The Km values at pH 7.5 for butyryl-CoA, acetoacetyl-CoA, and acetyl-CoA were 0.04, 1.10, and 3.33 mM, respectively. The Vmax values with butyryl-CoA and acetoacetyl-CoA were comparable, but the Vmax/Km was higher for butyryl-CoA than for acetoacetyl-CoA. An apparent Km of 6.5 mM for phosphate was obtained with butyryl-CoA as the cosubstrate, whereas it was 12.9 mM with acetoacetyl-CoA as the cosubstrate. It remains to be established whether the putative compound acetoacetyl phosphate is produced in the PTB-catalyzed reaction with acetoacetyl-CoA.

Full text

PDF
607

Images in this article

611Image
on p.611

Selected References

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

  1. BERGMEYER H. U., HOLZ G., KLOTZSCH H., LANG G. PHOSPHOTRANSACETYLASE AUS CLOSTRIDIUM KLUYVERI. ZUECHTUNG DES BACTERIUMS, ISOLIERUNG, KRISTALLISATION UND EIGENSCHAFTEN DES ENZYMS. Biochem Z. 1963;338:114–121. [PubMed] [Google Scholar]
  2. 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]
  3. Cary J. W., Petersen D. J., Papoutsakis E. T., Bennett G. N. Cloning and expression of Clostridium acetobutylicum phosphotransbutyrylase and butyrate kinase genes in Escherichia coli. J Bacteriol. 1988 Oct;170(10):4613–4618. doi: 10.1128/jb.170.10.4613-4618.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. ELLMAN G. L. Tissue sulfhydryl groups. Arch Biochem Biophys. 1959 May;82(1):70–77. doi: 10.1016/0003-9861(59)90090-6. [DOI] [PubMed] [Google Scholar]
  5. GAVARD R., HAUTECOEUR B., DESCOURTIEUX H. Phosphotransbutyrylase de Clostridium acetobutylicum. C R Hebd Seances Acad Sci. 1957 Apr 29;244(18):2323–2326. [PubMed] [Google Scholar]
  6. George H. A., Chen J. S. Acidic Conditions Are Not Obligatory for Onset of Butanol Formation by Clostridium beijerinckii (Synonym, C. butylicum). Appl Environ Microbiol. 1983 Aug;46(2):321–327. doi: 10.1128/aem.46.2.321-327.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hartmanis M. G. Butyrate kinase from Clostridium acetobutylicum. J Biol Chem. 1987 Jan 15;262(2):617–621. [PubMed] [Google Scholar]
  8. Hartmanis M. G., Gatenbeck S. Intermediary Metabolism in Clostridium acetobutylicum: Levels of Enzymes Involved in the Formation of Acetate and Butyrate. Appl Environ Microbiol. 1984 Jun;47(6):1277–1283. doi: 10.1128/aem.47.6.1277-1283.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Jones D. T., Woods D. R. Acetone-butanol fermentation revisited. Microbiol Rev. 1986 Dec;50(4):484–524. doi: 10.1128/mr.50.4.484-524.1986. [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. Robinson J. R., Sagers R. D. Phosphotransacetylase from Clostridium acidiurici. J Bacteriol. 1972 Oct;112(1):465–473. doi: 10.1128/jb.112.1.465-473.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. STERN J. R. Optical properties of aceto-acetyl-S-coenzyme A and its metal chelates. J Biol Chem. 1956 Jul;221(1):33–44. [PubMed] [Google Scholar]
  14. Sargent M. G. Fiftyfold amplification of the Lowry protein assay. Anal Biochem. 1987 Jun;163(2):476–481. doi: 10.1016/0003-2697(87)90251-x. [DOI] [PubMed] [Google Scholar]
  15. Senior P. J., Dawes E. A. The regulation of poly-beta-hydroxybutyrate metabolism in Azotobacter beijerinckii. Biochem J. 1973 May;134(1):225–238. doi: 10.1042/bj1340225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Srere P. A. Complexes of sequential metabolic enzymes. Annu Rev Biochem. 1987;56:89–124. doi: 10.1146/annurev.bi.56.070187.000513. [DOI] [PubMed] [Google Scholar]
  17. Terracciano J. S., Kashket E. R. Intracellular Conditions Required for Initiation of Solvent Production by Clostridium acetobutylicum. Appl Environ Microbiol. 1986 Jul;52(1):86–91. doi: 10.1128/aem.52.1.86-91.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Thauer R. K., Jungermann K., Decker K. Energy conservation in chemotrophic anaerobic bacteria. Bacteriol Rev. 1977 Mar;41(1):100–180. doi: 10.1128/br.41.1.100-180.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. VALENTINE R. C., WOLFE R. S. Purification and role of phosphotransbutyrylase. J Biol Chem. 1960 Jul;235:1948–1952. [PubMed] [Google Scholar]
  20. Wiesenborn D. P., Rudolph F. B., Papoutsakis E. T. Phosphotransbutyrylase from Clostridium acetobutylicum ATCC 824 and its role in acidogenesis. Appl Environ Microbiol. 1989 Feb;55(2):317–322. doi: 10.1128/aem.55.2.317-322.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Yan Run-Tao, Zhu Chang-Xi, Golemboski Christine, Chen Jiann-Shin. Expression of Solvent-Forming Enzymes and Onset of Solvent Production in Batch Cultures of Clostridium beijerinckii ("Clostridium butylicum"). Appl Environ Microbiol. 1988 Mar;54(3):642–648. doi: 10.1128/aem.54.3.642-648.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES