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. 1988 Nov;88(3):695–702. doi: 10.1104/pp.88.3.695

Oxygen-18 and Deuterium Labeling Studies of Choline Oxidation by Spinach and Sugar Beet 1

Claudia Lerma 1,2,2, Andrew D Hanson 1,2, David Rhodes 1,2
PMCID: PMC1055647  PMID: 16666370

Abstract

Chenopods synthesize betaine by a two-step oxidation of choline: choline → betaine aldehyde → betaine. The pathway is chloroplastic; the first step has been shown in isolated spinach (Spinacia oleracea L.) chloroplasts to be O2- and light-dependent, the role of light being to provide reducing power (P Weigel, EA Weretilnyk, AD Hanson 1988 Plant Physiol 86: 54-60). Here, we report use of in vivo18O- and 2H-labeling in conjunction with fast atom bombardment mass spectrometry to test for two hypothetical choline-oxidizing reactions that would explain the observed requirements for O2 and reductant: a desaturase or an oxygenase. Simple syntheses for 2H3-choline, 2H3, 18O-choline, and 2H3, 18O-betaine are given. A desaturase mechanism was sought by giving choline deuterated at the 2-carbon, or choline unlabeled at this position together with 2H2O and by analyzing newly synthesized betaine. About 15% of the 2H at C-2 was lost during oxidation of choline to betaine, and about 10% of the betaine made in the presence of 50% 2H2O was monodeuterated. These small effects are more consistent with chemical exchange than with a desaturase, because 10 to 15% losses of 2H from the C-2 position also occurred if choline was converted to betaine by a purified bacterial choline oxidase. To test for an oxygenase, the incorporation of 18O from 18O2 into newly synthesized betaine was compared with that from 18O-labeled choline, in light and darkness. Incorporation of 18O from 18O-choline was readily detectable and varied from about 15 to 50% of the theoretical maximum value; the 18O losses were attributable to exchange of the intermediate betaine aldehyde with water. In darkness, incorporation of 18O from 18O2 approached that from 18O-choline, but in the light was severalfold lower, presumably due to isotopic dilution by photosynthetic 16O2. These data indicate that the chloroplast choline-oxidizing enzyme is an oxygenase.

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Selected References

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

  1. Arakawa K., Takabe T., Sugiyama T., Akazawa T. Purification of betaine-aldehyde dehydrogenase from spinach leaves and preparation of its antibody. J Biochem. 1987 Jun;101(6):1485–1488. doi: 10.1093/oxfordjournals.jbchem.a122019. [DOI] [PubMed] [Google Scholar]
  2. Berry J. A., Osmond C. B., Lorimer G. H. Fixation of O(2) during Photorespiration: Kinetic and Steady-State Studies of the Photorespiratory Carbon Oxidation Cycle with Intact Leaves and Isolated Chloroplasts of C(3) Plants. Plant Physiol. 1978 Dec;62(6):954–967. doi: 10.1104/pp.62.6.954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bodley F. H., Blair A. H. Substrate characteristics of human liver aldehyde dehydrogenase. Can J Biochem. 1971 Jan;49(1):1–5. doi: 10.1139/o71-001. [DOI] [PubMed] [Google Scholar]
  4. GOOD N. E., BROWN A. H. The contribution of endogenous oxygen to the respiration of photosynthesizing Chlorella cells. Biochim Biophys Acta. 1961 Jul 8;50:544–554. doi: 10.1016/0006-3002(61)90014-2. [DOI] [PubMed] [Google Scholar]
  5. Hanson A. D., May A. M., Grumet R., Bode J., Jamieson G. C., Rhodes D. Betaine synthesis in chenopods: Localization in chloroplasts. Proc Natl Acad Sci U S A. 1985 Jun;82(11):3678–3682. doi: 10.1073/pnas.82.11.3678. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ikuta S., Imamura S., Misaki H., Horiuti Y. Purification and characterization of choline oxidase from Arthrobacter globiformis. J Biochem. 1977 Dec;82(6):1741–1749. doi: 10.1093/oxfordjournals.jbchem.a131872. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Matoh T., Watanabe J., Takahashi E. Sodium, Potassium, Chloride, and Betaine Concentrations in Isolated Vacuoles from Salt-Grown Atriplex gmelini Leaves. Plant Physiol. 1987 May;84(1):173–177. doi: 10.1104/pp.84.1.173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. McKeon T. A., Stumpf P. K. Purification and characterization of the stearoyl-acyl carrier protein desaturase and the acyl-acyl carrier protein thioesterase from maturing seeds of safflower. J Biol Chem. 1982 Oct 25;257(20):12141–12147. [PubMed] [Google Scholar]
  10. Naylor J. F., 3rd, Fridovich I. The form of the aldehyde susceptible to enzymic oxidation. J Biol Chem. 1968 Jan 25;243(2):341–345. [PubMed] [Google Scholar]
  11. Rhodes D., Rich P. J., Myers A. C., Reuter C. C., Jamieson G. C. Determination of Betaines by Fast Atom Bombardment Mass Spectrometry : Identification of Glycine Betaine Deficient Genotypes of Zea mays. Plant Physiol. 1987 Jul;84(3):781–788. doi: 10.1104/pp.84.3.781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Trentham D. R., McMurray C. H., Pogson C. I. The active chemical state of D-glyceraldehyde 3-phosphate in its reactions with D-glyceraldehyde 3-phosphate dehydrogenase, aldolase and triose phosphate isomerase. Biochem J. 1969 Aug;114(1):19–24. doi: 10.1042/bj1140019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Tsuge H., Nakano Y., Onishi H., Futamura Y., Ohashi K. A novel purification and some properties of rat liver mitochondrial choline dehydrogenase. Biochim Biophys Acta. 1980 Aug 7;614(2):274–284. doi: 10.1016/0005-2744(80)90217-x. [DOI] [PubMed] [Google Scholar]
  14. Weigel P., Lerma C., Hanson A. D. Choline oxidation by intact spinach chloroplasts. Plant Physiol. 1988 Jan;86(1):54–60. doi: 10.1104/pp.86.1.54. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Weigel P., Weretilnyk E. A., Hanson A. D. Betaine aldehyde oxidation by spinach chloroplasts. Plant Physiol. 1986 Nov;82(3):753–759. doi: 10.1104/pp.82.3.753. [DOI] [PMC free article] [PubMed] [Google Scholar]

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