accessible through a long, nonpolar tunnel lined with hydrophobic
residues that likely act as conduits for passage of the lipophilic
substrates. The role of ferrous iron in double bond cleaving
CCDs is to activate diatomic oxygen for cleavage of carotenoids.
The subsequent reaction follows a dioxygenase mechanism in
which both oxygen atoms are incorporated into the apocarotenoid
products [8].
The mammalian genome encodes three different types of
CCDs. The β-carotene-15,15
0 -dioxygenase (BCO1) and the
β-carotene-9
0 ,10
0 -dioxygenase (BCO2) enzymes catalyze oxidative
cleavage across double bonds at the C15, C15
0 and C9
0 , C10
0
positions on the carbon backbone of carotenoids, respectively
[9]. A third family member, the retinal pigment epithelium-specific
65 kDa protein (RPE65) does not possess the double bond cleavage activity of BCO1 and BCO2. It catalyzes both the ester cleavage and double bond isomerization reactions at position C10, C11
of retinyl esters (RE) into 11-cis-retinol [10].
For the biochemical characterization of mammalian BCO1 and
BCO2, E. coli expression systems have played a critical role. Initially, expression of these enzymes in carotenoid producing E. coli
strains provided a robust and reliable platform to biochemically
characterize these enzymes [9, 11]. The expression of BCO1
resulted in the conversion of the yellow β-carotene into colorless
retinoids. The color shift of the bacteria can be observed with the
naked eye and spectroscopically quantified. Additionally, methods
for the biochemical characterization of mammalian CCDs in cell
free systems were established. The challenging biochemistry of the
reactions catalyzed by CCDs initially led to conflicting results. For
instance, RPE65 was proposed to be a retinoid binding protein
[12, 13] and the existence of a vitamin A forming enzyme was even
disputed based on such assays [14]. Later, inconsistent results have
been published with regards to the biochemical activity of recombinant primate BCO2 [15–17]. Some of these discrepancies can be
explained by the demanding in vitro assay conditions for these
enzymes. CCDs catalyze reactions at the lipid aqueous interface
which poses a challenge with regards to substrate delivery to the
soluble enzymes. Also, chemical purity of the substrate is critical to
catalysis and to prevent misinterpretations due to nonenzymatic
oxidation of the substrates [14]. Moreover, heterologous expression of CCDs in E. coli expression system faces known obstacles
such as protein solubility and inclusion body formation [16]. This
chapter summarizes the advanced state of methodological knowledge about the biochemical characterization of recombinant mammalian BCO1 and BCO2. It provides a simple and efficient
protocol for the heterologous expression and biochemical characterization for this class of enzymes (Fig. 1) and discusses possible
pitfalls and problems.
76
Linda Dora Thomas et al.
residues that likely act as conduits for passage of the lipophilic
substrates. The role of ferrous iron in double bond cleaving
CCDs is to activate diatomic oxygen for cleavage of carotenoids.
The subsequent reaction follows a dioxygenase mechanism in
which both oxygen atoms are incorporated into the apocarotenoid
products [8].
The mammalian genome encodes three different types of
CCDs. The β-carotene-15,15
0 -dioxygenase (BCO1) and the
β-carotene-9
0 ,10
0 -dioxygenase (BCO2) enzymes catalyze oxidative
cleavage across double bonds at the C15, C15
0 and C9
0 , C10
0
positions on the carbon backbone of carotenoids, respectively
[9]. A third family member, the retinal pigment epithelium-specific
65 kDa protein (RPE65) does not possess the double bond cleavage activity of BCO1 and BCO2. It catalyzes both the ester cleavage and double bond isomerization reactions at position C10, C11
of retinyl esters (RE) into 11-cis-retinol [10].
For the biochemical characterization of mammalian BCO1 and
BCO2, E. coli expression systems have played a critical role. Initially, expression of these enzymes in carotenoid producing E. coli
strains provided a robust and reliable platform to biochemically
characterize these enzymes [9, 11]. The expression of BCO1
resulted in the conversion of the yellow β-carotene into colorless
retinoids. The color shift of the bacteria can be observed with the
naked eye and spectroscopically quantified. Additionally, methods
for the biochemical characterization of mammalian CCDs in cell
free systems were established. The challenging biochemistry of the
reactions catalyzed by CCDs initially led to conflicting results. For
instance, RPE65 was proposed to be a retinoid binding protein
[12, 13] and the existence of a vitamin A forming enzyme was even
disputed based on such assays [14]. Later, inconsistent results have
been published with regards to the biochemical activity of recombinant primate BCO2 [15–17]. Some of these discrepancies can be
explained by the demanding in vitro assay conditions for these
enzymes. CCDs catalyze reactions at the lipid aqueous interface
which poses a challenge with regards to substrate delivery to the
soluble enzymes. Also, chemical purity of the substrate is critical to
catalysis and to prevent misinterpretations due to nonenzymatic
oxidation of the substrates [14]. Moreover, heterologous expression of CCDs in E. coli expression system faces known obstacles
such as protein solubility and inclusion body formation [16]. This
chapter summarizes the advanced state of methodological knowledge about the biochemical characterization of recombinant mammalian BCO1 and BCO2. It provides a simple and efficient
protocol for the heterologous expression and biochemical characterization for this class of enzymes (Fig. 1) and discusses possible
pitfalls and problems.
76
Linda Dora Thomas et al.