Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2000 Dec 15;352(Pt 3):675–683.

Steady-state kinetic mechanism of the NADP+- and NAD+-dependent reactions catalysed by betaine aldehyde dehydrogenase from Pseudomonas aeruginosa.

R Velasco-García 1, L González-Segura 1, R A Muñoz-Clares 1
PMCID: PMC1221504  PMID: 11104673

Abstract

Betaine aldehyde dehydrogenase (BADH) catalyses the irreversible oxidation of betaine aldehyde to glycine betaine with the concomitant reduction of NAD(P)(+) to NADP(H). In Pseudomonas aeruginosa this reaction is a compulsory step in the assimilation of carbon and nitrogen when bacteria are growing in choline or choline precursors. The kinetic mechanisms of the NAD(+)- and NADP(+)-dependent reactions were examined by steady-state kinetic methods and by dinucleotide binding experiments. The double-reciprocal patterns obtained for initial velocity with NAD(P)(+) and for product and dead-end inhibition establish that both mechanisms are steady-state random. However, quantitative analysis of the inhibitions, and comparison with binding data, suggest a preferred route of addition of substrates and release of products in which NAD(P)(+) binds first and NAD(P)H leaves last, particularly in the NADP(+)-dependent reaction. Abortive binding of the dinucleotides, or their analogue ADP, in the betaine aldehyde site was inferred from total substrate inhibition by the dinucleotides, and parabolic inhibition by NADH and ADP. A weak partial uncompetitive substrate inhibition by the aldehyde was observed only in the NADP(+)-dependent reaction. The kinetics of P. aeruginosa BADH is very similar to that of glucose-6-phosphate dehydrogenase, suggesting that both enzymes fulfil a similar amphibolic metabolic role when the bacteria grow in choline and when they grow in glucose.

Full Text

The Full Text of this article is available as a PDF (175.6 KB).

Selected References

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

  1. Bar-Tana J., Cleland W. W. Rabbit muscle phosphofructokinase. II. Product and dead end inhibition. J Biol Chem. 1974 Feb 25;249(4):1271–1276. [PubMed] [Google Scholar]
  2. Bradbury S. L., Jakoby W. B. Ordered binding of substrates to yeast aldehyde dehydrogenase. J Biol Chem. 1971 Mar 25;246(6):1834–1840. [PubMed] [Google Scholar]
  3. CLELAND W. W. The kinetics of enzyme-catalyzed reactions with two or more substrates or products. I. Nomenclature and rate equations. Biochim Biophys Acta. 1963 Jan 8;67:104–137. doi: 10.1016/0006-3002(63)91800-6. [DOI] [PubMed] [Google Scholar]
  4. Chen R. F., Hayes J. E., Jr Fluorescence assay of high concentrations of DPNH and TPNH in a spectrophotofluorometer. Anal Biochem. 1965 Dec;13(3):523–529. doi: 10.1016/0003-2697(65)90347-7. [DOI] [PubMed] [Google Scholar]
  5. Chern M. K., Pietruszko R. Human aldehyde dehydrogenase E3 isozyme is a betaine aldehyde dehydrogenase. Biochem Biophys Res Commun. 1995 Aug 15;213(2):561–568. doi: 10.1006/bbrc.1995.2168. [DOI] [PubMed] [Google Scholar]
  6. D'Souza-Ault M. R., Smith L. T., Smith G. M. Roles of N-acetylglutaminylglutamine amide and glycine betaine in adaptation of Pseudomonas aeruginosa to osmotic stress. Appl Environ Microbiol. 1993 Feb;59(2):473–478. doi: 10.1128/aem.59.2.473-478.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Falkenberg P., Strøm A. R. Purification and characterization of osmoregulatory betaine aldehyde dehydrogenase of Escherichia coli. Biochim Biophys Acta. 1990 Jun 20;1034(3):253–259. doi: 10.1016/0304-4165(90)90046-y. [DOI] [PubMed] [Google Scholar]
  8. Farrés J., Wang T. T., Cunningham S. J., Weiner H. Investigation of the active site cysteine residue of rat liver mitochondrial aldehyde dehydrogenase by site-directed mutagenesis. Biochemistry. 1995 Feb 28;34(8):2592–2598. doi: 10.1021/bi00008a025. [DOI] [PubMed] [Google Scholar]
  9. Figueroa-Soto C. G., Valenzuela-Soto E. M. Kinetic study of porcine kidney betaine aldehyde dehydrogenase. Biochem Biophys Res Commun. 2000 Mar 16;269(2):596–603. doi: 10.1006/bbrc.2000.2337. [DOI] [PubMed] [Google Scholar]
  10. Forte-McRobbie C. M., Pietruszko R. Purification and characterization of human liver "high Km" aldehyde dehydrogenase and its identification as glutamic gamma-semialdehyde dehydrogenase. J Biol Chem. 1986 Feb 15;261(5):2154–2163. [PubMed] [Google Scholar]
  11. Hunt J. C., Phibbs P. V., Jr Regulation of alternate peripheral pathways of glucose catabolism during aerobic and anaerobic growth of Pseudomonas aeruginosa. J Bacteriol. 1983 May;154(2):793–802. doi: 10.1128/jb.154.2.793-802.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Johansson K., El-Ahmad M., Ramaswamy S., Hjelmqvist L., Jörnvall H., Eklund H. Structure of betaine aldehyde dehydrogenase at 2.1 A resolution. Protein Sci. 1998 Oct;7(10):2106–2117. doi: 10.1002/pro.5560071007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kilbourn J. P. Bacterial content and ionic composition of sputum in cystic fibrosis. Lancet. 1978 Feb 11;1(8059):334–334. doi: 10.1016/s0140-6736(78)90111-3. [DOI] [PubMed] [Google Scholar]
  14. Kutzenko A. S., Lamzin V. S., Popov V. O. Conserved supersecondary structural motif in NAD-dependent dehydrogenases. FEBS Lett. 1998 Feb 13;423(1):105–109. doi: 10.1016/s0014-5793(98)00074-x. [DOI] [PubMed] [Google Scholar]
  15. Landfald B., Strøm A. R. Choline-glycine betaine pathway confers a high level of osmotic tolerance in Escherichia coli. J Bacteriol. 1986 Mar;165(3):849–855. doi: 10.1128/jb.165.3.849-855.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Levy H. R., Christoff M., Ingulli J., Ho E. M. Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides: revised kinetic mechanism and kinetics of ATP inhibition. Arch Biochem Biophys. 1983 Apr 15;222(2):473–488. doi: 10.1016/0003-9861(83)90546-5. [DOI] [PubMed] [Google Scholar]
  17. Levy H. R., Daouk G. H., Katopes M. A. Regulation of coenzyme utilization by the dual nucleotide-specific glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroids. Arch Biochem Biophys. 1979 Dec;198(2):406–413. doi: 10.1016/0003-9861(79)90514-9. [DOI] [PubMed] [Google Scholar]
  18. Ma J. F., Hager P. W., Howell M. L., Phibbs P. V., Hassett D. J. Cloning and characterization of the Pseudomonas aeruginosa zwf gene encoding glucose-6-phosphate dehydrogenase, an enzyme important in resistance to methyl viologen (paraquat). J Bacteriol. 1998 Apr;180(7):1741–1749. doi: 10.1128/jb.180.7.1741-1749.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Muñoz-Clares R. A., Vojtechová M., Mújica-Jiménez C., Rodríguez-Sotres R. Effects of glycerol on the kinetic properties of betaine aldehyde dehydrogenase. Adv Exp Med Biol. 1997;414:261–268. doi: 10.1007/978-1-4615-5871-2_30. [DOI] [PubMed] [Google Scholar]
  20. Ostroff R. M., Vasil A. I., Vasil M. L. Molecular comparison of a nonhemolytic and a hemolytic phospholipase C from Pseudomonas aeruginosa. J Bacteriol. 1990 Oct;172(10):5915–5923. doi: 10.1128/jb.172.10.5915-5923.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pan S. M., Moreau R. A., Yu C., Huang A. H. Betaine accumulation and betaine-aldehyde dehydrogenase in spinach leaves. Plant Physiol. 1981 Jun;67(6):1105–1108. doi: 10.1104/pp.67.6.1105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Pesin S. R., Candia O. A. Acetylcholine concentration and its role in ionic transport by the corneal epithelium. Invest Ophthalmol Vis Sci. 1982 May;22(5):651–659. [PubMed] [Google Scholar]
  23. Petronini P. G., De Angelis E. M., Borghetti P., Borghetti A. F., Wheeler K. P. Modulation by betaine of cellular responses to osmotic stress. Biochem J. 1992 Feb 15;282(Pt 1):69–73. doi: 10.1042/bj2820069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rennick B. R. Renal tubule transport of organic cations. Am J Physiol. 1981 Feb;240(2):F83–F89. doi: 10.1152/ajprenal.1981.240.2.F83. [DOI] [PubMed] [Google Scholar]
  25. Rivett A. J., Tipton K. F. Kinetic studies with rat-brain succinic-semialdehyde dehydrogenase. Eur J Biochem. 1981 Jun;117(1):187–193. doi: 10.1111/j.1432-1033.1981.tb06319.x. [DOI] [PubMed] [Google Scholar]
  26. Ryzewski C. N., Pietruszko R. Kinetic mechanism of horse liver alcohol dehydrogenase SS. Biochemistry. 1980 Oct 14;19(21):4843–4848. doi: 10.1021/bi00562a021. [DOI] [PubMed] [Google Scholar]
  27. Sage A. E., Vasil A. I., Vasil M. L. Molecular characterization of mutants affected in the osmoprotectant-dependent induction of phospholipase C in Pseudomonas aeruginosa PAO1. Mol Microbiol. 1997 Jan;23(1):43–56. doi: 10.1046/j.1365-2958.1997.1681542.x. [DOI] [PubMed] [Google Scholar]
  28. Shortridge V. D., Lazdunski A., Vasil M. L. Osmoprotectants and phosphate regulate expression of phospholipase C in Pseudomonas aeruginosa. Mol Microbiol. 1992 Apr;6(7):863–871. doi: 10.1111/j.1365-2958.1992.tb01537.x. [DOI] [PubMed] [Google Scholar]
  29. Smith L. T., Pocard J. A., Bernard T., Le Rudulier D. Osmotic control of glycine betaine biosynthesis and degradation in Rhizobium meliloti. J Bacteriol. 1988 Jul;170(7):3142–3149. doi: 10.1128/jb.170.7.3142-3149.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Steinmetz C. G., Xie P., Weiner H., Hurley T. D. Structure of mitochondrial aldehyde dehydrogenase: the genetic component of ethanol aversion. Structure. 1997 May 15;5(5):701–711. doi: 10.1016/s0969-2126(97)00224-4. [DOI] [PubMed] [Google Scholar]
  31. Valenzuela-Soto E. M., Muñoz-Clares R. A. Betaine-aldehyde dehydrogenase from leaves of Amaranthus hypochondriacus L. exhibits an Iso Ordered Bi Bi steady state mechanism. J Biol Chem. 1993 Nov 15;268(32):23818–23823. [PubMed] [Google Scholar]
  32. Velasco-García R., Mújica-Jiménez C., Mendoza-Hernández G., Muñoz-Clares R. A. Rapid purification and properties of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa. J Bacteriol. 1999 Feb;181(4):1292–1300. doi: 10.1128/jb.181.4.1292-1300.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Vojtechová M., Rodríguez-Sotres R., Valenzuela-Soto E. M., Muñoz-Clares R. A. Substrate inhibition by betaine aldehyde of betaine aldehyde dehydrogenase from leaves of Amaranthus hypochondriacus L. Biochim Biophys Acta. 1997 Aug 15;1341(1):49–57. doi: 10.1016/s0167-4838(97)00059-9. [DOI] [PubMed] [Google Scholar]
  34. Weretilnyk E. A., Hanson A. D. Betaine aldehyde dehydrogenase from spinach leaves: purification, in vitro translation of the mRNA, and regulation by salinity. Arch Biochem Biophys. 1989 May 15;271(1):56–63. doi: 10.1016/0003-9861(89)90255-5. [DOI] [PubMed] [Google Scholar]
  35. Wright J. R., Clements J. A. Metabolism and turnover of lung surfactant. Am Rev Respir Dis. 1987 Aug;136(2):426–444. doi: 10.1164/ajrccm/136.2.426. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES