Skip to main content
NCBI home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1990 Sep;56(9):2591–2599. doi: 10.1128/aem.56.9.2591-2599.1990

Coenzyme A-acylating aldehyde dehydrogenase from Clostridium beijerinckii NRRL B592.

R T Yan 1, J S Chen 1
PMCID: PMC184801  PMID: 2275527

Abstract

Acetaldehyde and butyraldehyde are substrates for alcohol dehydrogenase in the production of ethanol and 1-butanol by solvent-producing clostridia. A coenzyme A (CoA)-acylating aldehyde dehydrogenase (ALDH), which also converts acyl-CoA to aldehyde and CoA, has been purified under anaerobic conditions from Clostridium beijerinckii NRRL B592. The ALDH showed a native molecular weight (Mr) of 100,000 and a subunit Mr of 55,000, suggesting that ALDH is dimeric. Purified ALDH contained no alcohol dehydrogenase activity. Activities measured with acetaldehyde and butyraldehyde as alternative substrates were copurified, indicating that the same ALDH can catalyze the formation of both aldehydes for ethanol and butanol production. Based on the Km and Vmax values for acetyl-CoA and butyryl-CoA, ALDH was more effective for the production of butyraldehyde than for acetaldehyde. ALDH could use either NAD(H) or NADP(H) as the coenzyme, but the Km for NAD(H) was much lower than that for NADP(H). Kinetic data suggest a ping-pong mechanism for the reaction. ALDH was more stable in Tris buffer than in phosphate buffer. The apparent optimum pH was between 6.5 and 7 for the forward reaction (the physiological direction; aldehyde forming), and it was 9.5 or higher for the reverse reaction (acyl-CoA forming). The ratio of NAD(H)/NADP(H)-linked activities increased with decreasing pH. ALDH was O2 sensitive, but it could be protected against O2 inactivation by dithiothreitol. The O2-inactivated enzyme could be reactivated by incubating the enzyme with CoA in the presence or absence of dithiothreitol prior to assay.

Full text

PDF
2591

Images in this article

2593Image
on p.2593

Selected References

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

  1. BURTON R. M., STADTMAN E. R. The oxidation of acetaldehyde to acetyl coenzyme A. J Biol Chem. 1953 Jun;202(2):873–890. [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. Byers D., Meighen E. Vibrio harveyi aldehyde dehydrogenase. Partial reversal of aldehyde oxidation and its possible role in the reduction of fatty acids for the bioluminescence reaction. J Biol Chem. 1984 Jun 10;259(11):7109–7114. [PubMed] [Google Scholar]
  4. CLELAND W. W. The kinetics of enzyme-catalyzed reactions with two or more substrates or products. III. Prediction of initial velocity and inhibition patterns by inspection. Biochim Biophys Acta. 1963 Feb 12;67:188–196. doi: 10.1016/0006-3002(63)91816-x. [DOI] [PubMed] [Google Scholar]
  5. Chen J. S., Blanchard D. K. A simple hydrogenase-linked assay for ferredoxin and flavodoxin. Anal Biochem. 1979 Feb;93(1):216–222. [PubMed] [Google Scholar]
  6. Cleland W. W. Statistical analysis of enzyme kinetic data. Methods Enzymol. 1979;63:103–138. doi: 10.1016/0076-6879(79)63008-2. [DOI] [PubMed] [Google Scholar]
  7. Cleland W. W. Substrate inhibition. Methods Enzymol. 1979;63:500–513. [PubMed] [Google Scholar]
  8. Griffith T. W., Sand D. M., Schlenk H. Reduction of fatty acids to alcohols in roe of gourami (Trichogaster cosby). Biochim Biophys Acta. 1981 Jul 24;665(1):34–39. doi: 10.1016/0005-2760(81)90228-9. [DOI] [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. Jones P. W., Turner J. M. A model for the common control of enzymes of ethanolamine catabolism in Escherichia coli. J Gen Microbiol. 1984 Apr;130(4):849–860. doi: 10.1099/00221287-130-4-849. [DOI] [PubMed] [Google Scholar]
  11. Palosaari N. R., Rogers P. Purification and properties of the inducible coenzyme A-linked butyraldehyde dehydrogenase from Clostridium acetobutylicum. J Bacteriol. 1988 Jul;170(7):2971–2976. doi: 10.1128/jb.170.7.2971-2976.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Patel R. N., Hou C. T., Derelanko P., Felix A. Purification and properties of a heme-containing aldehyde dehydrogenase from Methylosinus trichosporium. Arch Biochem Biophys. 1980 Sep;203(2):654–662. doi: 10.1016/0003-9861(80)90223-4. [DOI] [PubMed] [Google Scholar]
  13. Peterson J. B., LaRue T. A. Soluble aldehyde dehydrogenase and metabolism of aldehydes by soybean bacteroids. J Bacteriol. 1982 Sep;151(3):1473–1484. doi: 10.1128/jb.151.3.1473-1484.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Poels P. A., Groen B. W., Duine J. A. NAD(P)+-independent aldehyde dehydrogenase from Pseudomonas testosteroni. A novel type of molybdenum-containing hydroxylase. Eur J Biochem. 1987 Aug 3;166(3):575–579. doi: 10.1111/j.1432-1033.1987.tb13552.x. [DOI] [PubMed] [Google Scholar]
  15. Rudolph F. B., Purich D. L., Fromm H. J. Coenzyme A-linked aldehyde dehydrogenase from Escherichia coli. I. Partial purification, properties, and kinetic studies of the enzyme. J Biol Chem. 1968 Nov 10;243(21):5539–5545. [PubMed] [Google Scholar]
  16. Shone C. C., Fromm H. J. Steady-state and pre-steady-state kinetics of coenzyme A linked aldehyde dehydrogenase from Escherichia coli. Biochemistry. 1981 Dec 22;20(26):7494–7501. doi: 10.1021/bi00529a026. [DOI] [PubMed] [Google Scholar]
  17. Smith L. T., Kaplan N. O. Purification, properties, and kinetic mechanism of coenzyme A-linked aldehyde dehydrogenase from Clostridium kluyveri. Arch Biochem Biophys. 1980 Sep;203(2):663–675. doi: 10.1016/0003-9861(80)90224-6. [DOI] [PubMed] [Google Scholar]
  18. Steinman C. R., Jakoby W. B. Yeast aldehyde dehydrogenase. II. Properties of the homogeneous enzyme preparations. J Biol Chem. 1968 Feb 25;243(4):730–734. [PubMed] [Google Scholar]
  19. Thompson D. K., Chen J. S. Purification and properties of an acetoacetyl coenzyme A-reacting phosphotransbutyrylase from Clostridium beijerinckii ("Clostridium butylicum") NRRL B593. Appl Environ Microbiol. 1990 Mar;56(3):607–613. doi: 10.1128/aem.56.3.607-613.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Tipton K. F. Aldehyde dehydrogenases. Prog Clin Biol Res. 1985;174:3–13. [PubMed] [Google Scholar]
  21. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]
  22. Yan R. T., Zhu C. X., Chen J. S. Oxidation product(s) in acetaldehyde reacts with NAD(P)H and interferes with assay of alcohol dehydrogenase. Anal Biochem. 1987 Aug 1;164(2):362–366. doi: 10.1016/0003-2697(87)90505-7. [DOI] [PubMed] [Google Scholar]
  23. 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