Diverse Activities of Carotenoid Cleavage Oxygenases - Carotenoids: Physical, Chemical, and Biological Functions and Properties - page 402

OH

3

4
5

2
1

β

2'

1'

3'
4'

HO

α

6

6'

R 1

5'

NOV1
NOV2
SPA isoforms

R 2

OH

O
O

HO

R 1

Resveratrol : R 1 =OH R 2 =H

R 2

Piceatannol: R 1 =OH R 2 =OH

R 2 =OH

Rhapontigenin: R 1 = OCH 3

FIGURE 19.4 Cleavage of the a,b-double bond of stilbene substrates catalyzed by CCO homologs known as
LSDs. The four isoforms from Sphingomonas paucimobilis and two enzymes from Novophingobium aromati-
civoran s cleave stilbene substrates containing a 4′-oxygen functional group at one aromatic ring.

19.4 STRUCTURE AND MECHANISM OF CCOS
CCO enzymes are mononuclear, nonheme Fe 2+ enzymes. Enzymes with mononuclear, nonheme Fe 2+
centers in their active site are well-known for their catalytic versatility (reviewed by Straganz and
Nidetzky (2006)). Common reactions of these types of enzymes include carbon-oxygen bond forma-
tion and oxygenative bond cleavage. The predominant motif is a facial triad of two histidine ligands
and one carboxylate (reviewed by Hegg and Que [1997]). This motif paradigm can be found in many
mononuclear, nonheme Fe 2+ proteins with different structural folds (i.e., catechol 2,3-dioxygenase,
Rieske dioxygenase, pterin dioxygenase) (reviewed by Que and Ho [1996] and Costas et al. [2004}).
Alternative active site structures for mononuclear, nonheme Fe 2+ oxygenases exist. The majority
of these proteins possess the cupin superfamily fold (i.e., quercetin dioxygenase, cysteine dioxyge-
nase). The CCO family is unique among the mononuclear, nonheme Fe 2+ type oxygenases because
of its seven-bladed, b-propeller fold (Figure 19.5) (Kloer et al. 2005). The mononuclear iron in the
CCO active site also has a rare coordination geometry not found in other structurally characterized
dioxygenase enzymes. The iron is complexed by four histidines in an octahedral geometry with two
missing ligands, one of the coordination positions is occupied by water while the other position is
empty. The four metal-ligating histidine side chains are located at the propeller-axis with three of the
histidines held in place by hydrogen bonding to glutamate residues. All four histidines are perfectly
conserved among CCO family members. The rigid active site does not collapse or change shape
when iron is removed. Iron-free mutants are inactive and apoenzymes produced by chelation of the
iron cannot be reconstituted through the incorporation of other divalent metals (Poliakov et al. 2005,
Takahashi et al. 2005, Marasco et al. 2006).
So far, only one structure has been solved for a member of the CCO family. The structure of the
apocarotenoid cleavage enzyme SynACO from the cyanobacteria Synechocystis sp. PCC6803 was
solved to 2.4 Å (Kloer et al. 2005). The structure has a rare fold, consisting of a rigid, seven-bladed
propeller. Five of the blades are comprised of four antiparallel β-strands, and two blades have i ve
strands (Figure 19.5). The regions of highest conservation amongst the carotenoid oxygenases are
the β-strands of the propellers. The bottoms of the propellers are l at with the β-strands connected