Archives of Microbiology

, Volume 162, Issue 1–2, pp 48–56 | Cite as

Purification and characterization of a cytoplasmic enzyme component of the Na+-activated malonate decarboxylase system of Malonomonas rubra: acetyl-S-acyl carrier protein: malonate acyl carrier protein-SH transferase

  • Hubert Hilbi
  • Peter DimrothEmail author
Original Paper


Malonate decarboxylation by crude extracts of Malonomonas rubra was specifically activated by Na+ and less efficiently by Li+ ions. The extracts contained an enzyme catalyzing CoA transfer from malonyl-CoA to acetate, yielding acetyl-CoA and malonate. After about a 26-fold purification of the malonyl-CoA:acetate CoA transferase, an almost pure enzyme was obtained, indicating that about 4% of the cellular protein consisted of the CoA transferase. This abundance of the transferase is in accord with its proposed role as an enzyme component of the malonate decarboxylase system, the key enzyme of energy metabolism in this organism. The apparent molecular weight of the polypeptide was 67,000 as revealed from SDS-polyacrylamide gel electrophoresis. A similar molecular weight was estimated for the native transferase by gel chromatography, indicating that the enzyme exists as a monomer. Kinetic analyses of the CoA transferase yielded the following: pH-optimum at pH 5.5, an apparent Km for malonyl-CoA of 1.9mM, for acetate of 54mM, for acetyl-CoA of 6.9mM, and for malonate of 0.5mM. Malonate or citrate inhibited the enzyme with an apparent Ki of 0.4mM and 3.0mM, respectively. The isolated CoA transferase increased the activity of malonate decarboxylase of a crude enzyme system, in which part of the endogenous CoA transferase was inactivated by borohydride, about three-fold. These results indicate that the CoA transferase functions physiologically as a component of the malonate decarboxylase system, in which it catalyzes the transfer of acyl carrier protein from acetyl acyl carrier protein and malonate to yield malonyl acyl carrier protein and acetate. Malonate is thus activated on the enzyme by exchange for the catalytically important enzymebound acetyl thioester residues noted previously. This type of substrate activation resembles the catalytic mechanism of citrate lyase and citramalate lyase.

Key words

Malonyl-CoA:acetate CoA transferase Na+ transport decarboxylases Na+ cycle Citrate lyase Citramalate lyase CoA-like prosthetic group 


Citramalate lyase (EC Citrate lyase (EC Malonate decarboxylase (EC 4.1.1.-) 



5,5′ Dithiobis (2-nitrobenzoate)


2-(N-Morpholino)ethanesulfonic acid


N-[Tris(hydroxymethyl)-methyl]-3-aminopropanesulfonic acid


sodium dodecyl sulfate-polyacrylamide gel electrophoresis


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Buckel W, Bobi A (1976) The enzyme complex citramalate lyase from Clostridium tetanomorphum Eur J Biochem 64:255–262Google Scholar
  2. Buckel W, Semmler R (1982) A biotin-dependent sodium pump: glutaconyl-CoA decarboxylase from Acidaminococcus fermentans. FEBS Lett 148:35–38Google Scholar
  3. Buckel W, Dorn U, Semmler R (1981) Glutaconate CoA-transferase from Acidaminococcus fermentans. Eur J Biochem 118:315–321Google Scholar
  4. Dehning I, Schink B (1989) Malonomonas rubra gen. nov. sp. nov., a microaerotolerant anaerobic bacterium growing by decarboxylation of malonate. Arch Microbiol 151:427–433Google Scholar
  5. Dimroth P (1976) The prosthetic group of citrate-lyase acyl-carrier protein. Eur J Biochem 64:269–281Google Scholar
  6. Dimroth P (1987) Sodium ion transport decarboxylases and other aspects of sodium ion cyling in bacteria. Microbiol Rev 51: 320–340Google Scholar
  7. Dimroth P (1988) The role of vitamins and their carrier proteins in citrate fermentation. In: Kleinkauf H, Döhren H von, Jaenicke L (eds) The roots of modern biochemistry. de Gruyter, Berlin New York, pp 191–204Google Scholar
  8. Dimroth P, Eggerer H (1975) Evaluation of the protein components of citrate lyase from Klebsiella aerogenes Eur J Biochem 53:227–235Google Scholar
  9. Dimroth P, Loyal R (1977) Structure of the prosthetic groups of citrate lyase and citramalate lyase. FEBS Lett 76:280–283Google Scholar
  10. Dimroth P, Thomer A (1986) Kinetic analysis of the reaction mechanism of oxaloacetate decarboxylase from Klebsiella aerogenes. Eur J Biochem 156:157–162Google Scholar
  11. Dimroth P, Buckel W, Loyal R, Eggerer H (1977a) Isolation and function of the subunits of citramalate lyase and formation of hybrids with the subunits of citrate lyase. Eur J Biochem 80: 469–477Google Scholar
  12. Dimroth P, Loyal R, Eggerer H (1977b) Characterization of the isolated transferase subunit of citrate lyase as a CoA-transferase. Evidence against a covalent enzyme-substrate intermediate. Eur J Biochem 80:479–488Google Scholar
  13. Hilbi H, Dehning I, Schink B, Dimroth P (1992) Malonate decarboxylase of Malonomonas rubra, a novel type of biotin-containing acetyl enzyme. Eur J Biochem 207:117–123Google Scholar
  14. Hilbi H, Hermann R, Dimroth P (1993) The malonate decarboxylase enzyme system of Malonomonas rubra: evidence for the cytoplasmic location of the biotin-containing component. Arch Microbiol 160:126–131Google Scholar
  15. Hilpert W, Schink B, Dimroth P (1984) Life by a new decarboxylation-dependent energy conservation mechanism with Na+ as coupling ion. EMBO J 3:1665–1670Google Scholar
  16. Hoffmann A, Hilpert W, Dimroth P (1989) The carboxyltransferase activity of the sodium-ion-translocating methylmalonyl-CoA decarboxylase of Veillonella alcalescens Eur J Biochem 179:645–650Google Scholar
  17. Jencks WP (1973) Coenzyme A transferases. In: Boyer PD (ed) The enzymes 3rd edn, vol 9B. Academic Press, New York, pp 483–496Google Scholar
  18. King MT, Reiss PD (1985) Separation and measurement of shortchain coenzyme-A compounds in rat liver by reversed-phase high-performance liquid chromatography. Anal Biochem 146: 173–179Google Scholar
  19. Kluge C, Dimroth P (1992) Studies on Na+ and H+ translocation through the F0 part of the Na+-translocating F1F0 ATPase from Propionigenium modestum: discovery of a membrane potential dependent step. Biochemistry 31:12665–12672Google Scholar
  20. Robinson JB, Singh M, Srere PA (1976) Structure of the prosthetic group of Klebsiella aerogenes citrate (pro-3S)-lyase. Proc Natl Acad Sci USA 73:1872–1876Google Scholar
  21. Schmellenkamp H, Eggerer H (1974) Mechanism of enzymic acetylation of des-acetyl citrate lyase. Proc Natl Acad Sci USA 71:1987–1991Google Scholar
  22. Sramek SJ, Frerman FE (1975) Escherichia coli coenzyme A-transferase: kinetics, catalytic pathway and structure. Arch Biochem Biophys 171:27–35Google Scholar
  23. Tung KK, Wood WA (1975) Purification, new assay, and properties of coenzyme A transferase from Peptostreptococcus elsdenii. J Bacteriol 124:1462–1477Google Scholar

Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  1. 1.Mikrobiologisches InstitutEidgenössische Technische HochschuleZürichSwitzerland

Personalised recommendations