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Metabolism and Enzymology

Evidence for a Ferredoxin-Dependent Choline Monooxygenase from Spinach Chloroplast Stroma ¹

MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824, Department of Horticulture, Purdue University, West Lafayette, Indiana 47907, Department of Biology, University of Michigan, Ann Arbor, Michigan 48109, Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109

Chenopods synthesize betaine in the chloroplast via a two-step oxidation of choline: choline betaine aldehyde betaine. Our previous experiments with intact chloroplasts, and in vivo¹⁸O₂ labeling studies, led us to propose that the first step is mediated by a monooxygenase which uses photosynthetically generated reducing power (C Lerma, AD Hanson, D Rhodes [1988] Plant Physiol 88: 695-702). Here, we report the detection of such an activity in vitro. In the presence of O₂ and reduced ferredoxin, the stromal fraction from spinach (Spinacia oleracea) chloroplasts converted choline to betaine aldehyde at rates similar to those in intact chloroplasts (20-50 nanomoles per hour per milligram protein). Incorporation of ¹⁸O from ¹⁸O₂ by the in vitro reaction was demonstrated by fast atom bombardment mass spectrometry. Ferredoxin could be reduced either with thylakoids in the light, or with NADPH plus ferredoxin-NADP reductase in darkness; NADPH alone could not substitute for ferredoxin. No choline-oxidizing activity was detected in the stromal fraction of pea (Pisum sativum L.), a species that does not accumulate betaine. The spinach choline-oxidizing enzyme was stimulated by 10 millimolar Mg²⁺, had a pH optimum close to 8, and was insensitive to carbon monoxide. The specific activity was increased threefold in plants growing in 200 millimolar NaCl. Gel filtration experiments gave a molecular weight of 98 kilodaltons for the choline-oxidizing enzyme, and provided no evidence for other electron carriers which might mediate the reduction of the 98-kilodalton enzyme by ferredoxin.

³ Present address: Department of Biology, University College London, Gower Street, London WC1E 6BT, United Kingdom.

¹ Supported by the U.S. Department of Agriculture under grant 87-CRCR-1-2460 from the Competitive Grants Research Office, by the U.S. Department of Energy under contract DE-AC02-76ER0-1338, and by grants from the State of Michigan REF-Biotechnology Program, from CIBA-GEIGY Corporation, from the Beet Sugar Development Foundation, and from the Corporation for Science and Technology in Indiana. Michigan Agricultural Experiment Station journal article No. 12953. Purdue University Agricultural Experiment Station journal article No. 11804.

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