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

Abstract

Chenopods synthesize betaine in the chloroplast via a two-step oxidation of choline: choline .fwdarw. betaine aldehyde .fwdarw. betaine. Our previous experiments iwth intact chloroplasts, and in vivo 18O2 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 NADH plus ferredoxin-NADP reductase in darkness; NADH alone could not substitute for ferrodoxin. No choline-oxidizing acitivity 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 millometer 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 ferrodoxin.