Cloning and Expression of β,β-Carotene 15,15′-Dioxygenase

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We have identified from mouse the first mammalian ␤-carotene 15,15-dioxygenase (␤-CD), a crucial enzyme in development and metabolism that governs the de novo entry of vitamin A from plant-derived precursors. ␤-CD is related to the retinal pigment epithelium-expressed protein RPE65 and belongs to a diverse family that includes the plant 9-cis-epoxycarotenoid dioxygenase and bacterial lignostilbene dioxygenases. ␤-CD expression in Escherichia coli cells engineered to produce ␤-carotene led to the accumulation of all-trans-retinal at the expense of ␤-carotene, confirming that ␤-CD catalyzed the central cleavage of this vitamin A precursor. Purified recombinant ␤-CD protein cleaves ␤-carotene in vitro with a V max of 36 pmol of retinal/mg of enzyme/ min and a K m of 6 M. Non-provitamin A carotenoids were also cleaved, although with much lower activity. By Northern analysis, a 2.4-kilobase (kb) message was observed in liver, kidney, small intestine, and testis, tissues important in retinoid/carotenoid metabolism. This message encoded a 63-kDa cytosolic protein expressed in these tissues. A shorter transcript of 1.8 kb was found in testis and skin. Developmentally, the 2.4-kb mRNA was abundant at embryonic day 7, with lower expression at embryonic days 11, 13, and 15, suggesting a critical role for this enzyme in gastrulation. Identification of ␤-CD in an accessible model organism will create new opportunities to study vitamin A metabolism.In vertebrates, vitamin A in its various oxidative and isomeric forms is essential for embryonic development (1), pattern formation (2, 3), and vision (4). Retinoic acid, through its interaction with the nuclear retinoic acid receptor and retinoid X receptor, profoundly affects cell differentiation and development. Because animals are unable to synthesize vitamin A de novo from endogenous isoprenoid precursors, they must instead derive it from cleavage of ␤-carotene and certain other carotenoids with an unsubstituted ␤-ring (e.g. ␥-and ␣-carotenes, ␤-zeacarotene, and ␤-cryptoxanthin). It is generally accepted that central cleavage of ␤-carotene by a putative dioxygenase gives rise to two molecules of all-trans-retinal, whereas eccentric cleavage with subsequent processing leading to a single molecule of retinoic acid from an apocarotenal is quantitatively far less important (5). ␤-Carotene cleavage activity is reported highest in the intestinal mucosa, but is found at high activity levels in liver, kidney, lung, and fat tissues, among other sites. However, an inability to purify the protein catalyzing this reaction has hindered thorough investigation of this crucial first step in vitamin A metabolism.Because of a loose similarity between the mammalian protein RPE65 and the neoxanthin cleavage enzymes of plants, our laboratories have considered the hypothesis that the putative ␤-CD 1 would belong to an emerging family of carotenoidcleaving dioxygenases known mainly from examples in plants (6), but with members also in bacteria and Metazoa. The first described representative was a ba...

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confidence: 60%

Identification, Expression, and Substrate Specificity of a Mammalian β-Carotene 15,15′-Dioxygenase

Redmond

Gentleman

Duncan

et al. 2001

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225

“…For provitamin A carotenoids, such as ␤-carotene, ␣-carotene, and ␤-cryptoxanthin, central cleavage is a major pathway leading to vitamin A formation (2, 3). This pathway has been substantiated by the cloning of the central cleavage enzyme at their 15,15Ј-double bond, ␤-carotene 15,15Ј-monooxygenase (CMO1, 2 formerly called ␤-carotene 15,15Ј-dioxygenase) in different species (4,5), further classification of this enzyme as a non-heme iron monooxygenase (6), and recent biochemical and structural characterizations (7)(8)(9)(10)(11). Very recently, Poliakov et al (10) demonstrate that the four conserved histidines and one conserved glutamate are required for the iron coordination and catalytic activity of CMO1, which provided the first biochemical insight into the catalytic mechanism of CMO1.…”

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confidence: 99%

The Biochemical Characterization of Ferret Carotene-9′, 10′-Monooxygenase Catalyzing Cleavage of Carotenoids in Vitro and in Vivo

Ernst

et al. 2006

195

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Previous studies have shown that ␤-carotene 15,15-monooxygenase catalyzes the cleavage of ␤-carotene at the central carbon 15,15-double bond but cleaves lycopene with much lower activity. However, expressing the mouse carotene 9,10-monooxygenase (CMO2) in ␤-carotene/lycopene-synthesizing and -accumulating Escherichia coli strains leads to both a color shift and formation of apo-10-carotenoids, suggesting the oxidative cleavage of both carotenoids at their 9,10-double bond. Here we provide information on the biochemical characterization of CMO2 of the ferret, a model for human carotenoid metabolism, in terms of the kinetic analysis of ␤-carotene/lycopene cleavage into ␤-apo-10-carotenal/apo-10-lycopenal in vitro and the formation of apo-10-lycopenoids in ferrets in vivo. We demonstrate that the recombinant ferret CMO2 catalyzes the excentric cleavage of both all-trans-␤-carotene and the 5-cis-and 13-cis-isomers of lycopene at the 9,10-double bond but not all-trans-lycopene. The cleavage activity of ferret CMO2 was higher toward lycopene cis-isomers as compared with ␤-carotene as substrate. Iron was an essential co-factor for the reaction. Furthermore, all-trans-lycopene supplementation in ferrets resulted in significant accumulation of cis-isomers of lycopene and the formation of apo-10-lycopenol, as well as upregulation of the CMO2 expression in lung tissues. In addition, in vitro incubation of apo-10-lycopenal with the post-nuclear fraction of hepatic homogenates of ferrets resulted in the production of both apo-10-lycopenoic acid and apo-10-lycopenol, respectively, depending upon the presence of NAD ؉ or NADH as cofactors. Our finding of bioconversion of cis-isomers of lycopene into apo-10-lycopenoids by CMO2 is significant because cis-isomers of lycopene are a predominant form of lycopene in mammalian tissues and apo-lycopenoids may have specific biological activities related to human health.Carotenoids are lipophilic plant pigments with polyisoprenoid structures, typically containing a series of conjugated double bonds in the central chain of the molecule, which makes them susceptible to oxidative cleavage, and isomerization from trans to cis forms, with the formation of potentially bioactive metabolites. Knowledge of the biological effects of carotenoids, particularly for the impact of oxidation on these carotenoids and the potential for beneficial effects of small quantities or harmful effects of large quantities of the resulting metabolic products, has been reviewed recently (1). For provitamin A carotenoids, such as ␤-carotene, ␣-carotene, and ␤-cryptoxanthin, central cleavage is a major pathway leading to vitamin A formation (2, 3). This pathway has been substantiated by the cloning of the central cleavage enzyme at their 15,15Ј-double bond, ␤-carotene 15,15Ј-monooxygenase (CMO1, 2 formerly called ␤-carotene 15,15Ј-dioxygenase) in different species (4, 5), further classification of this enzyme as a non-heme iron monooxygenase (6), and recent biochemical and structural characterizations (7-11). Very rece...

“…In insects, the visual pigment retinal is formed by oxidative cleavage of ␤-carotene by ␤-carotene-15,15Ј-dioxygenase (4). Retinal is produced by an orthologous enzyme in vertebrates, where it is also converted to retinoic acid, a regulator of differentiation during embryogenesis (5). A distinct mammalian CCD is believed to cleave carotenoids asymmetrically at the 9,10 position (6) and, although its function is unclear, recent evidence suggests a role in the metabolism of dietary lycopene (7).…”

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confidence: 99%

Selective Inhibition of Carotenoid Cleavage Dioxygenases

Sergeant

Fox

et al. 2009

Members of the carotenoid cleavage dioxygenase family catalyze the oxidative cleavage of carotenoids at various chain positions, leading to the formation of a wide range of apocarotenoid signaling molecules. To explore the functions of this diverse enzyme family, we have used a chemical genetic approach to design selective inhibitors for different classes of carotenoid cleavage dioxygenase. A set of 18 arylalkyl-hydroxamic acids was synthesized in which the distance between an iron-chelating hydroxamic acid and an aromatic ring was varied; these compounds were screened as inhibitors of four different enzyme classes, either in vitro or in vivo. Potent inhibitors were found that selectively inhibited enzymes that cleave carotenoids at the 9,10 position; 50% inhibition was achieved at submicromolar concentrations. Application of certain inhibitors at 100 M to Arabidopsis node explants or whole plants led to increased shoot branching, consistent with inhibition of 9,10-cleavage.