Chapter 6
Expression and Characterization of Mammalian Carotenoid
Cleavage Dioxygenases
Linda Dora Thomas, Srinivasagan Ramkumar, and Johannes von Lintig
Abstract
Carotenoid cleavage dioxygenases (CCDs) are nonheme iron enzymes that catalyze double bond processing of carotenoids and their apocarotenoid metabolites. Mammalian genomes encode three members of
this protein family, namely BCO1, BCO2, and RPE65. Mutations and genetic polymorphism in the
corresponding genes are associated with inherited blinding diseases, vitamin A deficiency, and high
carotenoid plasma levels. Here we describe a method for the heterologous expression of mammalian
BCO1 and BCO2 in E. coli and the biochemical characterization of these recombinant enzymes. Dissecting
the enzymatic properties of CCDs will advance our knowledge of the biochemical processes that are govern
by these disease-associated enzymes and may assist the design of interventions directed against these disease
states.
Key words Carotenoids, Retinoids, Carotenoid cleavage dioxygenases, Protein expression, Enzyme
assays
1 Introduction
In mammals, carotenoids can be chemically converted to apocarotenoid metabolites [1]. These compounds include vitamin A aldehyde, from which all biologically active retinoids can be
synthesized, including visual chromophore and retinoic acid
[2]. Retinoic acid regulates gene expression throughout the mammalian life cycle and results in the effects of vitamin A in development, immunity, and cellular differentiation [3–5].
Enzymatic oxidative cleavage of carotenoids at a specific position of the polyene chain has been proposed as the method for
apocarotenoid production. This conversion is catalyzed by an
ancient family of carotenoid cleavage dioxygenases (CCDs)
[6]. CCDs are nonheme iron oxygenases with a characteristic tertiary structure comprised of a rigid seven-bladed β-propeller covered by a half-dome [7]. The ferrous iron in the active center is
Manuel Rodrı ´guez-Concepcio ´ n and Ralf Welsch (eds.), Plant and Food Carotenoids: Methods and Protocols,
Methods in Molecular Biology, vol. 2083, https://doi.org/10.1007/978-1-4939-9952-1_6,
© Springer Science+Business Media, LLC, part of Springer Nature 2020
75
Expression and Characterization of Mammalian Carotenoid
Cleavage Dioxygenases
Linda Dora Thomas, Srinivasagan Ramkumar, and Johannes von Lintig
Abstract
Carotenoid cleavage dioxygenases (CCDs) are nonheme iron enzymes that catalyze double bond processing of carotenoids and their apocarotenoid metabolites. Mammalian genomes encode three members of
this protein family, namely BCO1, BCO2, and RPE65. Mutations and genetic polymorphism in the
corresponding genes are associated with inherited blinding diseases, vitamin A deficiency, and high
carotenoid plasma levels. Here we describe a method for the heterologous expression of mammalian
BCO1 and BCO2 in E. coli and the biochemical characterization of these recombinant enzymes. Dissecting
the enzymatic properties of CCDs will advance our knowledge of the biochemical processes that are govern
by these disease-associated enzymes and may assist the design of interventions directed against these disease
states.
Key words Carotenoids, Retinoids, Carotenoid cleavage dioxygenases, Protein expression, Enzyme
assays
1 Introduction
In mammals, carotenoids can be chemically converted to apocarotenoid metabolites [1]. These compounds include vitamin A aldehyde, from which all biologically active retinoids can be
synthesized, including visual chromophore and retinoic acid
[2]. Retinoic acid regulates gene expression throughout the mammalian life cycle and results in the effects of vitamin A in development, immunity, and cellular differentiation [3–5].
Enzymatic oxidative cleavage of carotenoids at a specific position of the polyene chain has been proposed as the method for
apocarotenoid production. This conversion is catalyzed by an
ancient family of carotenoid cleavage dioxygenases (CCDs)
[6]. CCDs are nonheme iron oxygenases with a characteristic tertiary structure comprised of a rigid seven-bladed β-propeller covered by a half-dome [7]. The ferrous iron in the active center is
Manuel Rodrı ´guez-Concepcio ´ n and Ralf Welsch (eds.), Plant and Food Carotenoids: Methods and Protocols,
Methods in Molecular Biology, vol. 2083, https://doi.org/10.1007/978-1-4939-9952-1_6,
© Springer Science+Business Media, LLC, part of Springer Nature 2020
75