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

Abstract

Chenopods synthesize betaine in the chloroplast via a twostep oxidation of choline: choline betaine aldehyde betaine.Our previous experiments with intact chloroplasts, and in vivo 1802 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] insensitive to carbon monoxide. The specific activity was increased threefold in plants growing in 200 millimolar NaCI. 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.supports Wyn Jones's hypothesis (28) that betaine accumulation is a biochemical adaptation to salinity, with betaine acting as a major cytoplasmic osmolyte in salt-stressed plants (1 1, 17, 26). In stressed or unstressed plants, betaine is synthesized by a two-step oxidation of choline; both steps occur in the chloroplast in spinach and other chenopods (3):-2H -2H Choline -. betaine aldehyde -* betaine In spinach, the second step is catalyzed by a stromal BADH,4 which is encoded by a single nuclear gene (24,25). The enzyme for the first step is not yet known, although two lines of indirect evidence indicate that it is a monooxygenase (which could produce the hydrate form of betaine aldehyde by introducing an OH group at Cl of choline). First, in vivo labeling studies demonstrate that spinach leaf disks incorporate 180 from 1802 into betaine, and that this occurs at the choline -. betaine aldehyde step (9). Second, experiments with intact chloroplasts show that the choline -+ betaine aldehyde reaction requires photosynthetically generated reducing power as well as 02(23). The choline-oxidizing enzyme from spinach chloroplasts would therefore appear to be unlike the choline dehydrogenases (8,12,21)

Isolation of a choline monooxygenase cDNA clone from Amaranthus tricolor and its expressions under stress conditions

“…The quaternary structure of CMO is not clear. Spinach CMO might be a homodimer consisting of subunits around 43 kDa, but the possibility of additional subunits cannot be ruled out [7], [8]. Although the deduced amino acid sequence of A. tricolor CMO cDNA is similar to that of spinach in molecular weight, its subunit weighed less than spinach CMO subunit from this study (Fig 5).…”

“…CMO and BADH expressions in spinach leaves, and sugar beet leaves and taproots [4], [6], [7] were regulated by salt and drought stresses. A. caudatus L. in addition to Chenopodiaceae was first known to accumulate GB and express CMO protein under salt stress [5].…”

“…Afterward cDNAs encoding CMO proteins were cloned from both species [4], [6], and the deduced amino acid sequences showed 84% identity. Spinach CMO is a stromal, Fd-dependent monooxygenase with a Rieske-type [2Fe-2S] center [7], and is completely different from the bacterial choline dehydrogenase and oxidase enzymes. CMO was assumed to be unique among plant oxygenases [6], [8].…”

Synthesis of the osmoprotectant glycine betaine in Bacillus subtilis: characterization of the gbsAB genes

“…In Arthrobacter pascens, Arthrobacter globiformis, and an Alcaligenes sp., a bifunctional soluble choline oxidase that catalyzes the oxidation of choline and glycine betaine aldehyde to glycine betaine with the concomitant consumption of oxygen and the production of hydrogen peroxide has been found (23,40,47). In plants, glycine betaine synthesis occurs primarily in the chloroplasts and involves a soluble choline monooxygenase in combination with a soluble betaine aldehyde dehydrogenase (6,36,53). In a number of microorganisms and mammals, a membrane-bound choline dehydrogenase (which also can oxidize glycine betaine aldehyde to glycine betaine) and a soluble glycine betaine aldehyde dehydrogenase with a high substrate specificity are used for the synthesis of glycine betaine (33,38,46).…”

Choline oxidase, a catabolic enzyme in Arthrobacter pascens, facilitates adaptation to osmotic stress in Escherichia coli

“…In microorganisms and mammals a membrane-bound choline dehydrogenase (EC 1.1.99.1) is employed in conjunction with a soluble betaine aldehyde dehydrogenase (EC 1.2.1.8) (18,22,31,32,40). Plants utilize a soluble choline monooxygenase in combination with betaine aldehyde dehydrogenase (7,34). A third choline oxidation system, as yet found only in microorganisms, involves a soluble choline oxidase that is capable of catalyzing both reactions in vitro (20,33,49 (46), and pKK223-3 (6).…”