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Metabolism and Enzymology

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

MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824, Department of Horticulture, Purdue University, West Lafayette, Indiana 47907

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.

¹ Supported by the United States Department of Energy under contract DE-AC02-76ERO-1338, by the United States Department of Agriculture under grant 87-CRCR-1-2460 from the Competitive Grants Research Office, and by grants from the State of Michigan REED-Biotechnology Program, from CIBA-GEIGY Corporation, from the Beet Sugar Development Foundation, and from the Corporation for Science and Technology in Indiana. C. L. received a fellowship from CONACYT, Mexico. Michigan Agricultural Experiment journal article No. 12,633. Purdue University Agricultural Experiment Station journal article No. 11,624.

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