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in ferrets that encodes a protein of 540 amino acids and has a 82% identity with human carotene,
9
-oxygenase. Further analysis revealed that the enzyme is expressed in the testis, the liver, the
lung, the prostate, the intestine, the stomach, and the kidneys of ferrets, similar to the expression
pattern of human CMO2 (Lindqvist et al. 2005). Using the recombinant ferret CMO2 expressed in
Spodoptera frugiperda
(Sf9) insect cells for kinetic analysis, we found that the cleavage of carotenoids
by the ferret CMO2 occurs in a pH-, incubation time-, protein dose-, and substrate dose-dependant
manner (Hu et al. 2006). Notably, the optimum pH for CMO2 is 8.5, which differs from the opti-
mum pH (7.7) for the activity of CMO1, the central cleavage enzyme for carotenoids (Lindqvist and
Andersson 2002). The difference in optimum pH between these two carotenoid cleavage enzymes
may indicate different roles of the two pathways in carotenoid metabolism or different functions in
various pathophysiological conditions, which need further investigation. Nonetheless, similar to the
CMO1, we found that the cleavage activity of ferret CMO2 for both b-carotene and lycopene was
iron-dependent, indicating that iron is an essential cofactor for the enzymatic cleavage activity of
carotenoids. This is supported by the existence of four conserved histidines residues in the ferret
CMO2 (Hu et al. 2006). These data are in agreement with previous observations demonstrating that
these conserved histidines act as putative iron-binding residues for iron coordination in apocarote-
noid 15,15
′
,10
′
-oxygenase (Kloer et al. 2005) and CMO1 (Poliakov et al. 2005) supporting the notion that
the entire superfamily of oxygenases shares a common structure (Poliakov et al. 2005).
Interestingly, we demonstrated that the recombinant ferret CMO2 catalyzes the excentric cleav-
age of all-
trans
b-carotene and
cis
-lycopene isomers effectively but not all-
trans
lycopene at the
9
′
double bond (Hu et al. 2006). While we estimated a
K
m
of 3.5 mM for all-
trans
b-carotene
based on the CMO2 expressed in SF9 cells, we could not calculate the kinetic constants of CMO2
for lycopene due to difi culty in controlling auto-isomerization, thus, necessitating the use of mixed
isomers of lycopene as the substrates for kinetic analysis. Since the lycopene substrate mixture
contains only ~20% as
cis
isomers and considering that the ferret CMO2 would not cleave all-
trans
lycopene, we speculate that the
K
m
for
cis
-lycopene is actually much lower than that of the lyco-
pene isomer mixture. This indicates that
cis
-lycopene may act as a better substrate than all-
trans
b-carotene for the ferret CMO2. The mechanism whereby ferret CMO2 preferentially cleaves the
5-
cis
and 13-
cis
-isomers of lycopene into apo-10
′
,10
′
-lycopenal but not all-
trans
lycopene is currently
unknown. One possible explanation is that the chemical structure of
cis
isomers of lycopene could
mimic the ring structure of the b-carotene molecule and i t into the substrate-enzyme binding
pocket (Figure 20.1). Although this hypothesis warrants further investigation, the observation that
the supplementation of all-
trans
lycopene results in a signii cant increase in
cis
-lycopene tissue
concentration in ferrets underlies the signii cance of this observation (Boileau et al. 1999, Liu et al.
2003, 2006).
′
20.2.2.3 Regulation of Carotene Oxidases
A number of animal studies have demonstrated that CMO1 activity is affected by nutritional sta-
tus, such as vitamin A status (Parvin and Sivakumar 2000, van Vliet et al. 1996). Other studies
have indicated that the expression of CMO1 may be regulated at the transcriptional level through
feedback regulatory mechanisms via interactions between retinoic acid and its nuclear recep-
tors (Bachmann et al. 2002, Chichili et al. 2005). Recent molecular studies of the mouse and the
human CMO1 promoters demonstrated the presence of a peroxisome proliferator response ele-
ment (PPRE) (Boulanger et al. 2003, Gong et al. 2006). PPARg (peroxisome proliferators acti-
vated receptor-g) and RXRa (retinoid X repetor-a) agonists were shown to transactivate the CMO1
promoter-reporter when cotransfected with the corresponding nuclear receptor (Boulanger et al.
2003). The analysis of the human CMO1 promoter identii ed an additional enhancer element. A
myocyte enhancer factor-2 (MEF2) binding site was identii ed and when mutated reduced luciferase
activity by ~30% (Gong et al. 2006). The
in vivo
importance of the MEF2 binding site is not fully
understood. Nonetheless, the regulation by PPAR and RXR indicates a regulatory link between
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