Diverse Activities of Carotenoid Cleavage Oxygenases - Carotenoids: Physical, Chemical, and Biological Functions and Properties - page 400

19.3.4 C YANOBACTERIAL CCO S
Environmental sampling and observations identii ed apocarotenoid products from microbial sources
including cyanobacteria. Cyanobacteria have been found to excrete apocarotenoids such as methyl
ketones, trans -geranyl acetone, and ionone derivatives (Enzell 1985). C 13 and C 9 volatiles, b-ionone
and b-cyclocitral, respectively, were isolated from Microcystis aeruginosa and Anabaena cylindrical
(Juttner 1976). In follow-up studies, an enzyme from Microcystis PCC7806 was found that specii -
cally cleaves b-carotene and zeaxanthin asymmetrically at the 7,8 (7 )-bonds to form volatile aroma

compounds (b-cyclocitral, hydroxyl-b-cyclocitral) and crocetindial (8,8

′ ,8 ′

dial).
The enzyme did not cleave the major cyanobacterial carotenoids echinenone or myxoxanthophyll.
In the native host, this enzyme was associated with the membrane, required iron, and is sensitive to
sulfhydryl reagents, antioxidants, and chelating agents (Juttner 1985). Labeling studies coni rmed
that at least one of the aldehyde oxygens of the cleavage products is derived from dioxygen. This
enzyme is the i rst microbial CCO enzyme described, but it has not yet been cloned.
The proliferation of microbial genome sequencing projects has made the identii cation of CCO
homologs in microorganisms possible. Genome mining based on protein sequence homology has
resulted in the cloning of several microbial CCOs (Marasco et al. 2006) (Figure 19.2 and Table 19.4).
The i rst report of a cloned bacterial CCO was in 2005 for an apocarotenoid cleavage enzyme
from Synechocystis PCC 6803 (SynACO) (Ruch et al. 2005). This enzyme was crystallized and
the structure solved at 2.4 Å resolution (PDB code 2BIX; see below) (Kloer et al. 2005). In vitro
assays testing apocarotenoids of various chain lengths, b-ring substitutions, and alcohol deriva-
tives showed cleavage of the 15,15

′ -diapocarotene-8,8 ′

carotenal (C 30 ) was converted
into retinal (C 20 ) (Figure 19.1) and related apocarotenoid compounds 10

′ bond of apocarotenoids. b-apo-8 ′

′ apocarotenal (C 27 ) and

3-hydroxy-b-apo-12 carotenal were also substrates. Despite reacting with a variety of carotenoid

chain lengths and both aldehydes and alcohols, SynACO does not cleave C 25 , C 35 or full-length caro-
tenoids. Regardless of the substrate, the 15,15

′

-bond was always the target suggesting the b-ionone
ring is the determining factor in cleavage site specii city (Ruch et al. 2005). Cleavage specii city
by a related enzyme from Nostoc sp. PCC 7120 (NosACO or NSC2; 53% identity) showed similar
specii city (Scherzinger et al. 2006). Kinetic analysis showed higher binding afi nity for aldehydes,
but alcohols and unsubstituted rings were converted more quickly by NSC2 (Scherzinger et al.
2006). Based on modeling NSC2 on the SynACO scaffold, Scherzinger et al. (2006) predicted that
the enzyme will not cleave full-length carotenoids (see below). However, b-carotene cleavage was
observed by Marasco et al. (2006). The slow turnover of full-length carotenoids by the microbial
CCO enzymes may account for this contradiction.
The i lamentous, diazotrophic Nostoc sp. PCC7120 genome contains three open reading frames
with homology to CCOs. Cloning and partial characterization of these enzymes identii ed three
distinct cleavage activities (Marasco et al. 2006). NSC1 ( all1106 ) cleaves full-length carotenoids
and b-apo-8

′

apocarotenal in the 9,10-position resulting in b-ionone. NSC3 ( all4895 ) was found to
cleave the 9,10-bond of apocarotenoids only. As previously mentioned, NSC2 ( all4284 ) cleaved the
15,15

′

-bond to form retinal. Included in the study was a survey of four other cyanobacterial genomes
and related open reading frames ( Nostoc punctiforme , Synechocystis sp. PCC6803, Synechococcus
elongatus PCC7942, Prochlorococcus marinus MIT9313). The unicellular Synechococcus elon-
gatus PCC7942 enzyme (SYO) bleached carotenoid containing E. coli in a similar manner to
NSC 1 and 2 (Marasco et al. 2006). Further studies have indicated cleavage of the 15,15

′

′ -bond

of b-carotene, zeaxanthin, and b-apo-8 carotenal (Marasco and Schmidt-Dannert, unpublished).

The other i lamentous cyanobacteria, Nostoc punctiforme, contained 4 CCO paralogs, two of which
were found to cleave apocarotenoids and the other two were inactive against (apo)carotenoid sub-
strates tested. NOP1 (Npun1384) cleaved 9,10-position of b-apo-8

′

′ carotenal and NOP4 (Npun5487)

cleaved the 15,15 -bond to form retinal; NOP2 (Npun0528) and NOP3 (Npun6913) were inactive

(Marasco and Schmidt-Dannert, unpublished). The putative CCO homolog from Prochlorococcus

′