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reactive double bond near the Fe
2+
and dioxygen coordinates in a side-on fashion and displaces both
water molecules to form a ternary complex (Kloer et al. 2005). At this point, the reaction can pro-
ceed via two possible intermediates (either a dioxetane or epoxide intermediate). Both intermediates
would be expected to decompose into the same cleavage product (Kloer and Schulz 2006).
The enzyme mechanism is known to consume dioxygen, but whether this enzyme catalyzes
oxidative cleavage via a mono- or dioxygenase mechanism cannot be deduced from the structure.
Both dioxygenase and monooxygenase mechanisms have been hypothesized based on conl icting
18
O labeling studies (Figure 19.6). Incorporation of both oxygen atoms of O
2
are supported by
studies from
Arabidopsis thaliana
producing b-ionone (Schmidt et al. 2006) and plants producing
ABA (Zeevaart et al. 1989). In contrast, the formation of retinal has been suggested at different
times with different enzyme examples both as a dioxygenase mechanism and a monooxygenase-
like mechanism through a postulated epoxy intermediate (Leuenberger et al. 2001). Labeling
studies with
Microcystis
utilizing whole cells under an
18
O
2
atmosphere showed an
18
O label
on b-cyclocitral (86%), hydroxyl-b-cyclocitral (20.5% labeled), while the dialdehyde cleavage
product crocetindial (8,8
dial) was unlabeled. The authors suggest the lack
of labeling on the linear cleavage product was due to a high exchange rate of the labeled oxygen
with water. The exchange rate of the aldehyde oxygen in this study was indeed high (33% after
20 h). When the cells were exposed to H
2
18
O in the converse labeling experiment, the b-cyclocitral
was labeled at 17%. The authors suggest a dioxygenase mechanism from this evidence. Studies
with endogenous 15,15
′
-diapocarotene-8,8
′
CCO enzymes from chicken mucosa using both
17
O and H
2
18
O showed
incorporation of one oxygen from O
2
and one from water through a proposed epoxide intermedi-
ate (Leuenberger et al. 2001). In this study, an equal enrichment of
17
O and
18
O (52%:41%) was
observed. This study has been criticized, however, for the long incubation time and the coupling
of the enzyme assay with the horse liver alcohol dehydrogenase enzyme to reduce the aldehyde
to an alcohol (Schmidt et al. 2006). Very recently, labeling studies using H
2
18
O and
18
O
2
with the
stilbene cleaving enzymes NOV1 show that at least these enzymes cleave the interphenyl dou-
ble bond of stilbenes with a monooxygenase mechanism (Marasco and Schmidt-Dannert 2008).
Unlike carotenoid cleavage by CCOs, stilbene cleavage catalyzed by NOV2 is relatively fast,
stilbenes are readily solubilized in the assays and cleavage products can be rapidly isolated and
detected by GC-MS, which reduces the extent of unspecii c label exchange which is a problem in
studies with these enzymes.
The current controversy over the oxygenase mechanism of this family of nonheme iron enzymes
stems from contradictory i ndings from labeling studies and a lack of rigorous biophysical studies
(Leuenberger et al. 2001, Schmidt et al. 2006). The poor activities of recombinant CCOs in
in vitro
assays and cleavage of water insoluble substrates may largely be responsible for the lack of rigorous
mechanistic studies of this class of nonheme iron oxygenases. To date there has been no cofactor
identii ed that is associated with the cleavage activity of the CCOs. Given the poor reactivity
in
vitro
, it is plausible that there is a nontraditional cofactor associated with the enzyme (Paik et al.
2001). Another possibility is that the membrane association of the enzymes limits their activities
unless when incorporated in liposomes. As more enzyme examples are discovered and better reac-
tion conditions developed, more careful biochemical characterization may be possible.
′
19.5 BIOLOGICAL FUNCTIONS OF APOCAROTENOIDS
The structural variety of apocarotenoids results in divergent biological functions. To date, the known
biological activities of apocarotenoids generated by plant and microbial CCOs are more diverse
than those produced in animals. Pigmentation is the most obvious function of apocarotenoids in
plants; some cleavage products maintain an extended conjugated system and serve as pigments
(e.g., saffron). When acting as pigments in plant tissues, they are often found in specialized plastids
known as chromoplasts. In thylakoid membranes, apocarotenoids act as accessory pigments and
are involved in photoprotection; smaller cleavage compounds protect against UVB by absorbing
between 280 and 320 nm.
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