Oxygen-18 and deuterium labeling studies of choline oxidation by spinach and sugar beet

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 O(2)- 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 vivo(18)O- and (2)H-labeling in conjunction with fast atom bombardment mass spectrometry to test for two hypothetical choline-oxidizing reactions that would explain the observed requirements for O(2) and reductant: a desaturase or an oxygenase. Simple syntheses for (2)H(3)-choline, (2)H(3), (18)O-choline, and (2)H(3), (18)O-betaine are given. A desaturase mechanism was sought by giving choline deuterated at the 2-carbon, or choline unlabeled at this position together with (2)H(2)O and by analyzing newly synthesized betaine. About 15% of the (2)H at C-2 was lost during oxidation of choline to betaine, and about 10% of the betaine made in the presence of 50% (2)H(2)O was monodeuterated. These small effects are more consistent with chemical exchange than with a desaturase, because 10 to 15% losses of (2)H 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 (18)O from (18)O(2) into newly synthesized betaine was compared with that from (18)O-labeled choline, in light and darkness. Incorporation of (18)O from (18)O-choline was readily detectable and varied from about 15 to 50% of the theoretical maximum value; the (18)O losses were attributable to exchange of the intermediate betaine aldehyde with water. In darkness, incorporation of (18)O from (18)O(2) approached that from (18)O-choline, but in the light was severalfold lower, presumably due to isotopic dilution by photosynthetic (16)O(2). These data indicate that the chloroplast choline-oxidizing enzyme is an oxygenase.