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

Abstract

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...

Optimized Formation of Detergent Micelles of β-Carotene and Retinal Production Using Recombinant Human β,β-Carotene 15,15'-Monooxygenase

“…These micelles are solubilized under aqueous conditions and are absorbed by intestinal cells (1)(2)(3)(4)(5)(6). Moreover, detergent micelles of -carotene have been used in the conversion of -carotene to retinal under aqueous conditions (4,10,(14)(15)(16)(17)(18)(19)(20). Thus, we investigated the effect of factors such as solvent, detergent, and substrate on the formation of detergent micelles of -carotene for retinal production under aqueous conditions.…”

“…However, because these micelles were used to investigate enzyme characterization other than retinal production, only a small amount of -carotene was used (4,10,(14)(15)(16)(17)(18)(19)(20).…”

Phytoene, phytofluene, and lycopene from tomato powder differentially accumulate in tissues of male Fisher 344 rats

“…Both CMO I and CMO II are highly expressed in mouse and human liver [41,42]. Indeed, LYC metabolites, including apo-8′-lycopenal and apo-10′-lycopenal, are in the liver of LYC-fed rats and ferrets, respectively, suggesting LYC cleavage by CMO II [43,44]. Therefore, if PE and PF have less affinity for CMO I and II, then enzymatic and non-enzymatic cleavage of LYC in the liver would ultimately result in decreased hepatic LYC concentrations relative to PE and PF.…”

NinaB combines carotenoid oxygenase and retinoid isomerase activity in a single polypeptide

“…Thereafter, several related carotenoid-15,15Ј-oxygenases (CMO1) have been identified and characterized in vertebrates including human beings (4)(5)(6)(7). In addition, in vertebrates a second type of carotenoid-oxygenase, carotenoid-9Ј,10Ј-monooxygenase (CMO2) has been identified that cleaves carotenoids asymmetrically (8,9), although its physiological function is not yet fully understood.…”

“…Interestingly, carotenoid-oxygenases and the retinoid isomerase RPE65 belong to a family of structurally related nonheme iron oxygenases (9,14) that was first described in plants (15). Whereas mammalian genomes encode three family members (RPE65, CMO1 and CMO2), insect genomes (such as Drosophila melanogaster, Anopheles gambiae, or Apis mellifera) encode only a single member of this class of enzyme, namely NinaB.…”