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Originally published In Press as doi:10.1074/jbc.M606299200 on August 18, 2006

J. Biol. Chem., Vol. 281, Issue 42, 31583-31593, October 20, 2006
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Identification of Carotenoid Cleavage Dioxygenases from Nostoc sp. PCC 7120 with Different Cleavage Activities*Formula

Erin K. Marasco1, Kimleng Vay, and Claudia Schmidt-Dannert2

From the Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, St. Paul, Minnesota 55108

Received for publication, June 30, 2006 , and in revised form, July 31, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Carotenoid cleavage dioxygenases (CCDs) are a class of enzymes that oxidatively cleave carotenoids into apocarotenoids. Dioxygenases have been identified in plants and animals and produce a wide variety of cleavage products. Despite what is known about apocarotenoids in higher organisms, very little is known about apocarotenoids and CCDs in microorganisms. This study surveyed cleavage activities of ten putative carotenoid cleavage dioxygenases from five different cyanobacteria in recombinant Escherichia coli cells producing different carotenoid substrates. Three CCD homologs identified in Nostoc sp. PCC 7120 were purified, and their cleavage activities were investigated. Two of the three enzymes showed cleavage of beta,beta-carotene at the 9,10 and 15,15' positions, respectively. The third enzyme did not cleave full-length carotenoids but cleaved the apocarotenoid beta-apo-8'-carotenal at the 9,10 position. 9,10-Apocarotenoid cleavage specificity has previously not been described. The diversity of carotenoid cleavage activities identified in one cyanobacteria suggests that CCDs not only facilitate the degradation of photosynthetic pigments but generate apocarotenals with yet to be determined biological roles in microorganisms.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Oxidative cleavage of carotenoids results in a structurally diverse class of compounds known as apocarotenoids. Apocarotenoids are generated when a double bond in the carotenoid backbone is cleaved with molecular oxygen forming an aldehyde or ketone at the cleavage site. All conjugated double bonds that compose carotenoid backbones are potential cleavage sites, leading to a wide array of apocarotenoid products. Apocarotenoids can be generated by nonspecific mechanisms via lipoxygenases, peroxidases, and photooxidation (1), but a major contributor to the formation of biologically active compounds is a class of non-heme iron enzymes known as carotenoid cleavage dioxygenases (CCDs).3 Until recently there was controversy as to whether the mechanism of cleavage was mono- or dioxygenase, but based on recent evidence they will be referred to as dioxygenases in this report (2).

CCDs produce biologically active apocarotenoids in plants and animals that have several major functions, including the formation of signaling molecules in plants, ecologically important volatile aroma and flavor compounds, and the formation of retinoids with developmental and physiological functions (3-6). The first carotenoid cleavage enzyme cloned was a nine-cis-epoxy dioxygenase (NCED) Vp14 from Zea mays, which is involved in the biosynthesis of abscisic acid in plants (7). Based on sequence homology, similar enzymes were discovered in other plants and animals involved in the biosynthesis of phytohormones, aroma compounds, and vitamin A (8-14). Several different types of carotenoid cleavage dioxygenases can be distinguished based on their specific cleavage sites and/or carotenoid substrates (3). NCEDs are 11,12-double bond-specific plant dioxygenases (7, 15). Other plant carotenoid dioxygenases that cleave all-trans carotenoids cleave asymmetrically the 9,10 bond (12) or symmetrically the 9,10(9',10') bonds (13, 16) or 7,8(7',8') (10) and 5,6 bonds (9). The predominant class of dioxygenases found in metazoans cleave at the 15,15' double bond to form two molecules of retinal (17-23) (Fig. 1A).

Despite the increasing number of carotenoid cleavage dioxygenases characterized from plants and animals over the last few years, relatively little is known about the function of the dioxygenases in microorganisms. Two known examples in the literature describe cleavage of substrates with conjugated backbones similar to carotenoids but do not report the cleavage of full-length carotenoids. One enzyme from Sphingomonas paucimobilis was characterized as a lignostilbene dioxygenase, because it catalyzes analogous central double bond cleavage of the bicyclic 4,4'-dihydroxy-3,3-dimethoxystilbene to form two molecules of vanillin (24-27). The second enzyme is from Synechocystis PCC 6803 and cleaves apocarotenoids such as beta-apo-8'-carotenal at the 15,15 double bond into retinal (28). The crystal structure of this enzyme was also solved and showed a Fe2+ 4-histidine arrangement in the axis of a seven-bladed beta-propeller (29). The role of retinal in microorganisms is not clear, although homologous dioxygenases in vertebrates produce retinal and retinoic acids as signaling compounds in development as well as in visual pigments (11, 14, 19, 20, 22, 30). Recent discoveries of eubacterial sensory rhodopsins provide a potential function for retinal as a chromophore component (31).

In this study we sought to survey putative carotenoid cleavage dioxygenases identified in five sequenced cyanobacterial strains. As photosynthetic organisms, cyanobacteria synthesize carotenoids and cleavage enzymes may play an important role in regulating carotenoid levels during photosynthesis or, as in plants, cleave carotenoids into signaling molecules (12). Three of the five cyanobacterial strains surveyed each have two to four predicted carotenoid dioxygenase paralogs suggesting that the enzymes may have different functions. We cloned ten predicted carotenoid dioxygenases, including the described enzyme from Synechocystis PCC 6803, and surveyed their in vivo carotenoid cleavage activities by expressing the genes in five recombinant Escherichia coli strains producing different carotenoid substrates. Several enzymes in this initial screen showed activity against multiple tested carotenoid substrates. Three enzymes from Nostoc sp. PCC 7120, two of which were among the most active in the in vivo survey, were selected for a more detailed characterization of their cleavage activities. In this study we report on the presence of multiple dioxygenases in a microbial strain with different cleavage specificities for both full-length and apocarotenoids.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals—The following chemicals were used: beta-ionone (Sigma-Aldrich), beta,beta-carotene (Sigma-Aldrich), beta-apo-8'-carotenal (Fluka), and all-trans-retinal (Sigma). All-trans-retinal was reduced to all-trans-retinol with NaBH4 (32). All solvents were of HPLC grade and purchased through Fisher Scientific (Pittsburgh, PA). HPLC grade water was purchased from Mallinckrodt Chemicals (Phillipsburg, NJ). Vent DNA polymerase, T4 DNA ligase, and restriction enzymes were from New England Biolabs (Boston, MA). Restriction buffers were SuRE/Cut buffers from Roche Applied Science.

Gene Cloning—Homology searches were performed using NCBI BLAST software based on the Crocus sativa 7,8(7',8') zeaxanthin carotenoid dioxygenase protein (CAD33262 [GenBank] ) and a putative CCD homolog sequence from Nostoc punctiforme (ZP_00106997). CCD homologs were identified in cyanobacterial genome sequences obtained from NCBI and the Joint Genome Institute. Identified cyanobacterial genes were amplified from genomic DNA isolated from cyanobacterial strains by PCR with Vent polymerase using gene-specific primers with added restriction sites (supplemental Table S1). Known CCDs from Arabidopsis thaliana AtCCD1 (13) and Mus musculus MmBCO (21) were amplified from cDNA. A. thaliana cDNA was prepared as described previously (33) and mouse cDNA from CD1 mouse liver (Charles River Laboratory, Wilmington, MA) was a generous gift from the Marker laboratory (University of Minnesota, Minneapolis, MN). The obtained PCR products were digested with their respective restriction enzymes, gel-purified, and ligated into the constitutive expression vector pUCmod for in vivo analysis of cleavage activities (34). Cloned gene sequences were verified by sequencing.

For protein overexpression, CCD genes were subcloned into the NdeI and XhoI sites of the inducible expression vector pET20b+ (Invitrogen) using gene-specific primers containing the corresponding restriction sites. The stop codon was eliminated from the sequences for in-frame fusion with a C-terminal 6x histidine tag encoded on the pET20b+ vector to facilitate protein purification.

Culture Conditions and Strains—All cloning and investigations of in vivo carotenoid cleavage activities were carried out in E. coli strain JM109. E. coli cultures were grown in Luria-Bertani (LB) medium supplemented with appropriate antibiotics carbenicillin or ampicillin (100 µgml-1), and chloramphenicol (50 µgml-1), as indicated, at 30 °C. E. coli strain BL21 was used for protein expression. Cyanobacterial strains used were obtained from ATCC (Nostoc sp. PCC 7120 (27893), Synechocystis sp. PCC 6803 (27184), and Synechococcus sp. PCC 7942 (33912)) and grown using culture conditions recommended by the supplier. Genomic DNA was purchased from ATCC for N. punctiforme PCC 73102 or provided by the Chisholm Laboratory for Prochlorococcus marinus MIT9313 (Boston, MA).

In Vivo Analysis of Carotenoid Cleavage Activity—Putative dioxygenase enzymes on pUCmod were co-expressed with carotenoid biosynthetic genes expressed from pACmod in E. coli JM109 (supplemental Table S1). The construction of carotenoid biosynthesis pathways on the pAC plasmid has been described previously (34-36). Briefly, beta,beta-carotene biosynthetic genes were expressed from plasmid pAC-crtE-crtB-crtI14-crtY, zeaxanthin genes from plasmid pAC-crtE-crtB-crtI14-crtY-crtX, lycopene genes from plasmid pAC-crtE-crtB-crtI, torulene genes from plasmid pAC-crtE-crtB-crtI14-crtY2, and diapocarotendial genes from plasmid pAC-ispA-crtM-crtN-crtOx (supplemental Table S1). The latter plasmid was constructed from pAC-ispA-crtM-crtN (35) by insertion of the crtOx expression cassette from pUC-crtOx (36) in a manner similar to that described for the other pAC constructs (35).

To investigate in vivo carotenoid cleavage, single colonies of E. coli JM109 transformants harboring the carotenoid and dioxygenase plasmids to be tested were grown overnight and for 48 h in 4 ml of LB media supplemented with carbenicillin and chloramphenicol at 30 °C. The color intensities of the resulting cell pellets from six replicate cultures of each carotenoid and dioxygenase combination were then compared by visible inspection to control cells harboring the corresponding carotenoid plasmid and empty pUCmod plasmid. Cell pellets with visible bleaching observed in 4-ml cultures were confirmed with larger 50-ml overnight cultures.

Analysis of carotenoid accumulation and apocarotenoid cleavage products in E. coli cells co-expressing carotenoid and dioxygenase genes was performed by organic extraction of cells from 100-ml overnight cultures grown in LB essentially as described in Schwartz et al. (12). In brief, for total carotenoid quantification cultures were centrifuged, and the pellets were resuspended in 1 ml of 33% formaldehyde solution. Then 10 ml of ethanol was added, and the samples were placed in a sonicating water bath at 4 °C for 3 h. After this incubation, cell debris was centrifuged and the absorbance of the extract was measured at 450 nm by a spectrophotometer, and concentration was calculated using an extinction coefficient of 14,040 m-1 cm-1 for beta,beta-carotene (32). For HPLC analysis of carotenoids, cell pellets were extracted three times with acetone, and the extracts were combined and dried under nitrogen. Samples were stored at -20 °C until HPLC analysis (see below). For apocarotenoid analysis by HPLC, cell pellets were sequentially extracted with formaldehyde solution, methanol, and diethyl ether. The apocarotenoid-containing diethyl ether layer was reduced with nitrogen and stored at -20 °C until analyzed by HPLC (see below).

Protein Expression and Purification—The pET20b+ plasmid containing CCD genes was used for overexpression of CCD proteins in E. coli BL21. Recombinant E. coli BL21 cells were co-transformed with different chaperone plasmids from Takara (Madison, WI) (Supplementary Table 1) to optimize soluble protein expression. E. coli BL21 co-transformed with dioxygenase and different combinations of chaperone genes (dnaK-dnaJ-grpE-groES-groES, groES-groEL, dnaK-dnaJ-grpE, groES-groEL-tig, and tig) were grown overnight at 30 °C in 5 ml of LB media. This culture was used to inoculate (1:100) 500 ml of LB, and chaperone expression was induced with 0.5 mg/ml arabinose or 5 ng/ml tetracycline as indicated. The cells were grown at 30 °C until an optical density of A0.6 was reached. Then the cultures were cooled on ice and induced with 1 mM isopropyl-beta-D-thiogalactopyranoside before cultivation was continued overnight at 18 °C, and cells were harvested by centrifugation at 4000 rpm. Cell pellets were resuspended in 4 ml of phosphate buffer (50 mM NaPO4, pH 7.0) and lysed by sonication (Branson, Danbury, CT) using a 5-min cycle with 30 s on/45 s off. Following centrifugation, samples representing total cell protein, insoluble, and soluble protein were subjected to SDS-PAGE analysis to identify chaperones that produced the most soluble dioxygenase protein. Following these experiments, all subsequent dioxygenase protein expressions were done with the pGro7 plasmid containing the genes for GroEL and GroES using the above conditions. Cleared cell lysates from recombinant E. coli BL21 cells were used for in vitro assays and protein purification.

Dioxygenase proteins were purified using Talon resin immobilized metal affinity chromatography (Invitrogen). Soluble protein was loaded onto the column and eluted in 50 mM phosphate buffer, pH 7.2, with 300 mM imidazole following three washing steps. The protein eluted in a 4-ml fraction and was concentrated using an Amicon ultracentrifuge concentrator with a 10,000 molecular weight cut-off. The Amicon concentrator was used to desalt the protein by exchanging the buffer four times against 50 mM phosphate buffer, pH 7.2. Proteins were >90% pure as judged by SDS gels stained by Coomassie Blue dye. Protein concentrations were determined using Bradford reagent (Bio-Rad). Elemental analysis of nitric acid digests of purified Nostoc sp. PCC 7120 proteins after reconstitution in assay buffer was conducted by inductively coupled plasma mass spectrometry (Elan 5000, PerkinElmer Life Sciences).

In Vitro Assays—In vitro reactions were performed as in Schwartz et al. (13) and Ruch (28) with some modifications. Assays with whole cell lysate were performed with 100 µl of whole cell lysate added to 200 µl of 50 mM Tris-HCl, pH 7.0, buffer (with 300 mM NaCl, 10 mM dithiothreitol, 10 mM sodium ascorbate, 0.5 mM FeSO4, 0.05% Triton X-100 final concentration). The buffer and lysate equilibrated for 20 min before the addition of 10 µl of 1 mM substrate (beta,beta-carotene or beta-apo-8'-carotenal) in methanol. Assays with purified proteins were performed with 100-250 µg of purified NSC proteins in 300-µl reactions containing 50 mM phosphate buffer, pH 7.2 (containing 300 mM NaCl, 0.5 mM FeSO4, 50 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)). After 20 min of equilibration, 40 µl of 2 mM substrate (beta,beta-carotene or beta-apo-8'-carotenal) was added in 1% Tween 40.

In vitro reactions were carried out at 30 °C for the prescribed amount of time (2-12 h) before being quenched with 50 µl of 33% formaldehyde solution for 10 min at 37 °C. After the samples were quenched with formaldehyde, 1 ml of acetonitrile was added and the organic layer saved for analysis by HPLC. Alternatively, samples were extracted twice with chloroform to recover small amounts of cleavage products. It should be noted that trace amounts of acid present in chloroform lead to increased isomerization of double bonds present in carotenoid compounds (32) and thus, splitting of product peaks into multiple isomer peaks in HPLC chromatograms.

Time-course assays were carried out using 100 µg of purified NSC1 protein in a 300-µl reactions. Samples were monitored on the spectrophotometer every hour by a wavelength scan from 350 to 650 nm before being extracted. At set time points, 0, 2, 3, 4, 5, and 14 h a time-point sample was removed and extracted, and the organic fraction was saved for analysis by HPLC.

HPLC and LC/MS Analysis—HPLC analysis was performed using an 1100 HPLC system equipped with a photodiode array detector (Agilent, Palo Alto, CA). Several HPLC conditions were utilized to determine different carotenoid cleavage products. Retinal was detected by applying 50 µl of sample to an Adsorbosil C-18 column (4.6 x 250 mm, 5 µm, Alltech, Deer-field, IL). The gradient program was modified from Ruch et al. (28) using a solvent system of MeOH:tert-butylmethyl ether: water (120:4:40, v/v) (B) and MeOH:tert-butylmethyl ether (500:500 v/v) (A). The gradient conditions were 100% solvent B to 43% solvent B over 45 min, 43% solvent B to 0% solvent B for 11 min, and 0% solvent B for 14 min with a flow rate of 1 ml min-1. Retinal compounds were identified by comparisons of retention times and UV-visible spectra of standard compounds and mass spectrometry (see below). Dialdehyde products were determined by applying 100 µl of sample to a Zorbax RX-C18 column (4.6 x 250 mm, 5 µm, Agilent). The solvent system was MeOH:water (70:30, v/v) with 0.1% ammonium acetate (B) and MeOH (A). The gradient conditions were 100% solvent B to 0% solvent B over 16 min, 0% solvent B until 26 min, and then return to 100% A with a flow rate of 0.8 ml min-1. Gradients were extended for 2-3 min to better resolve minor cleavage products after complete conversion of beta-apo-8'-carotenal. Organic extracts from E. coli pellets (40 µl) were applied to a Zorbax SB-C18 column (4.6 x 250 mm, 5 µm, Agilent) and eluted under isocratic conditions with a solvent system containing 90% acetonitrile and 10% water with 0.1% ammonium acetate with a flow rate of 0.8 ml min-1 (37).

Mass fragmentation spectra were monitored in a mass range of m/z 100-800 on an LCQ mass spectrophotometer equipped with an atmosphere pressure chemical ionization interface (Thermo Finnigan). The chromatography conditions were identical to the HPLC conditions described above.

GC/MS Analysis—GC/MS analysis was carried out on a Varian 3800 GC coupled to an ion-trap mass spectrometer (Saturn, Palo Alto, CA). Separation was carried out on a DB5 capillary column (30 m x 0.25 mm inner diameter x 1.5 µm) with an injection port temperature of 230 °C and helium as a carrier gas. Mass spectra were recorded in electron impact ionization mode. Samples were analyzed by solid phase micro-extraction on a 100-µm polydimethylsiloxane fiber from Supelco (Belle-fonte, PA). Volatiles were absorbed from the headspace over 15 min, and the fiber was desorbed in the injection port for 5 min. The temperature program was 100 °C for 5 min with an increase of 8 °C/min to a final oven temperature of 290 °C. Mass spectra were scanned in the range of m/z 50-300 atomic mass units at 1-s intervals. Linear retention time and electron impact ionization mass spectrum data of the beta-ionone cleavage product was compared with an authentic standard.


Figure 1
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  		FIGURE 1.
Characterized and predicted cyanobacterial carotenoid cleavage dioxygenases investigated in this study. A, cleavage activities reported for carotenoid dioxygenases. NSC1 and NSC3 (this study) cleave beta-apo-8'-carotenal at the 9,10 double bond forming 8',10-apocarotenal and beta-ionone. NSC2 (this study) cleaves beta-apo-8'-carotenal at the 15,15' double bond forming retinal. B, unrooted phylogenetic tree of select carotenoid dioxygenases and their cleavage activities (if known). Alignments were created, and the tree was computed using ClustalX. Full-length sequences for the following genes were used: S. paucimobilis sp. (SPA), AAB35856; Nostoc sp. PCC 7120 (NSC1), BAB73063; N. punctiforme (NOP1), ZP_00106997; Arabidopsis thaliana (AtCCD1), NP_191911.1; Crocus sativus (CsCCD), AJ132927; Bixa orellana (BoLCD), AJ489277; Crocus sativus (CsZCD), AJ489276; Z. mays (VP14), AAB62181.1; Arabidopsis thaliana (AtNCED2), NP_193569.1; P. marinus MIT 9313 (PRO1), ZP_00114109; Synechocystis PCC 6803 (SYC2), S76169; Synechococcus elongates sp. PCC 7942 (SYO1), AAC12875; Nostoc sp. PCC 7120 (NSC2), AE2341; N. punctiforme (NOP3), ZP_00112423; Synechocystis PCC 6803 (SYC1), S76206; Nostoc sp. PCC 7120 (NSC3), BAB76594; N. punctiforme (NOP4), ZP_00111018; Mus musculus (MmBCOI), Q9JJS6; M. musculus (MmBCOII), Q99NF1; and M. musculus (MmRPE65), Q91ZQ5.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Cyanobacterial Carotenoid Cleavage Enzyme Homologs Vary in Function—Cyanobacterial homologs of eukaryotic CCDs are present in all sequenced cyanobacterial genomes, but to date only one from Synechocystis PCC 6803 (SYC2) has been characterized (28). Predicted CCD homologs are present in Nostoc sp. PCC 7120 (NSC1, NSC2, and NSC3), N. punctiforme (NOP1, NOP2, NOP3, and NOP4), P. marinus MIT9313 (PRO1), Synechocystis PCC 6803 (SYC1 and SYC2), and Synechococcus elongates PCC 7942 (SYO1). Most cyanobacterial strains, except for P. marinus MIT9313 and S. elongates PCC 7942, have several predicted CCD paralogs. A sequence alignment of predicted cyanobacterial CCDs with select characterized eukaryotic CCDs reveals clustering of proteins onto distinct branches of a phylogenetic tree that may reflect different activities (Fig. 1B). Carotenoid cleavage enzymes have different cleavage site specificities, but currently there is no obvious sequence information that can be used to predict their cleavage activities. Sequence similarity among dioxygenases ranges from 74% identity (NSC1 to NOP1) to 27% identity (NSC1 to SYC1). Interestingly, sequence identity among cyanobacterial CCDs is lower than the identity between some plant and cyanobacterial dioxygenases. For example, NSC1 shares 44% identity with a CCD from Arabidopsis (AtCCD1) but only 26% identity to another predicted dioxygenase paralog (NSC3) in its own genome. All characterized and predicted CCDs contain four conserved histidine residues, which coordinate the non-heme active site iron (supplemental Fig. S1) (29). We confirmed this for NSC1 from Nostoc sp. PCC 7120 by replacing each individual histidine with an alanine (H68A, H173A, H221A, and H285A), which, as expected, resulted in inactive proteins (data not shown). Similar loss of activity has been reported for histidine mutants of the mouse 15,15' oxygenase MmBCOI and MmRPE65 (38, 39). With the exception of the metal center in the active site, specific regions that confer cleavage activities have not yet been identified, although a recent crystal structure begins to address this question (29).

Co-expression of Cyanobacterial Dioxygenases in Carotenoid-accumulating E. coli—To determine the activity of the predicted cyanobacterial carotenoid cleavage enzymes, genes were amplified from genomic DNA and cloned into pUCmod, a plasmid with an optimized ribosomal binding site and a constitutive lac-promoter for constitutive protein expression (34). One of the predicted CCD sequences (NOP2) could not be amplified from genomic DNA using primer sequences derived from the genomic sequences in the NCBI data base, although paralogs from the same organism were readily amplified and cloned. Expression levels in E. coli of the cloned cyanobacterial CCD homologs (except NSC1) and two known CCDs (see below) cloned as controls were comparable at 5-10% of the total protein. NSC1 was overexpressed at three times the level of all the other proteins.

In previous research, we have created recombinant E. coli cells that co-express different combinations of carotenoid biosynthetic genes, directing the synthesis of diverse carotenoids compounds in this non-carotenogenic host (34-36). E. coli strains accumulating different carotenoids substrates were co-transformed with the cloned cyanobacterial CCD homologs and with two known CCD genes (encoding the 9,10,9',10' enzyme from Arabidopsis (AtCCD) (13) and 15,15' enzyme from mouse MmBCOI (14) as controls). Cleavage of carotenoids in E. coli destroys the chromophore of carotenoids, causing a loss of cell color (also referred to as bleaching).

We selected carotenoid substrates representing different structural features for this initial survey of in vivo cleavage activity (Table 1). Both lycopene, a linear carotenoid with a backbone of 40 carbons, and beta,beta-carotene, a bicyclic carotenoid of the same length, have previously been shown to be substrates for carotenoid cleavage enzymes (Fig. 1B). Torulene represents a monocyclic carotenoid of 40 carbons. Zeaxanthin is derived from beta-carotene by the addition of 4'-hydroxyl groups on both beta-ionone rings. The importance of carotenoid chain length was tested by including in this survey a novel linear carotenoid of 30 carbon atoms (36). Phylogenetic tree analysis (Fig. 1B) and alignments (supplemental Fig. S1) showed higher similarity between predicted cyanobacterial enzymes and plant CCDs rather than NCEDs, so xanthophylls with cis-double bonds were excluded from this study. In addition to monitoring the color phenotypes of CCD-expressing E. coli cultures, carotenoid levels were monitored through extraction and spectrophotometric quantification (12).


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  		TABLE 1
Survey of cleavage activities of predicted cyanobacterial carotenoid cleavage dioxygenases

Carotenoid cleaving enzymes were co-transformed into E. coli cells expressing different C40 and C30 carotenoid biosynthetic pathways. Bleaching of cell color was compared to control cells without and with known cleavage enzymes AtCCD1 (9,10,9',10' CCD from Arabidopsis (13)) and MmBCO (15,15' CCD from mouse (21)) after 12-h growth in LB at 30 °C. n/e, was not evaluated; 0-5, indicates color loss (0) being no visible loss of color, and substantial bleaching (5). Residual carotenoids extracted from cells co-expressing the {gamma},{gamma}-carotene pathway andNSCor control cleavage enzyme, AtCCD1, were quantified and compared to control cells without cleavage enzymes. Data represent the average of three extractions from 50-ml cultures grown for 48 h in LB at 30 °C.

 
In vivo results showed varying bleaching efficiencies for the predicted cyanobacterial CCDs in different E. coli carotenoid backgrounds (Table 1). The two most active CCDs (NSC1 and NSC2) resulted in less pigment accumulation when expressed in cells producing lycopene, beta,beta-carotene, zeaxanthin, torulene, or C30 dialdehyde. NSC1, NSC2, and SYO showed distinctive bleaching with a range of linear and cyclic carotenoid substrates. In contrast, carotenoid accumulation was only slightly inhibited in NOP1, NOP3, and PROI co-transformants, and there was no observable bleaching for NSC3, NOP4, SYC1, and SYC2 or the empty vector control. The 9,10-cleavage enzyme from Arabidopsis AtCCD1 showed greater bleaching activity than all of the cyanobacterial dioxygenases tested, irrespective of the carotenoid substrate. This may indicate that the substrates tested were not the natural or optimal substrate for the microbial dioxygenases. Alternatively, the localization of AtCCD in E. coli may be different than that of microbial CCDs affecting substrate accessibility. The second control enzyme, MmBCOI, included in this study displayed a distinct specificity for beta,beta-carotene (14). Accumulation of linear carotenoids was affected to a greater extent than cyclized carotenoids by NSC2, suggesting that NSC2 has higher preference for linear carotenoids than either AtCCD1 or NSC1. In addition to common carotenoids, the dioxygenases were co-expressed in E. coli cells producing an unusual acyclic xanthophyll, 4,4'-diapocarotene-4,4'-dial (36). Loss of color with this carotenoid background was observed for AtCCD1 as well as NSC1, NSC2, and SYO. From these results it appears that NSC1, NSC2, and SYO cleave full-length carotenoids, whereas the other cyanobacterial CCDs either do not cleave full-length carotenoids (as e.g. reported for the apocarotenoid cleavage dioxygenase SYC2 (28)) or are not functional under the conditions tested (e.g. SYC1 was previously reported to be inactive (28)).


Figure 2
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  		FIGURE 2.
SDS gel of NSC1 expression in E. coli cells stained by Coomassie Blue. Cells were grown overnight at 20 °C with or without chaperones co-expressed and lysed by sonication. Lane 1, pET-NSC1 total protein uninduced; lane 2, pET-NSC1 total protein induced; lane 3, pET-NSC1 soluble fraction induced; lane 4, pET-NSC1 plus pGRO7 uninduced; lane 5, pET-NSC1 plus pGRO7 total protein induced; and lane 6, pET20b+_NSC1 plus pGRO7 soluble fraction. The arrow points to the NSC1 protein. The pGRO7 plasmid (Takara) expresses groES-groEL (10 and 60 kDa).

 
The finding that two of the three cyanobacterial CCDs with in vivo activity against full-length carotenoids come from one strain, Nostoc sp. PCC 7120, led us to further characterize the cleavage activities of all three NSC enzymes and determine whether they evolved different cleavage functions as their divergent sequences may suggest (Fig. 1B).

Identification of NSC in Vivo Cleavage Products—Color bleaching of cells in vivo indicated that full-length carotenoids were being cleaved by NSC1 and NSC2, but not by NSC3 (Table 1). This was confirmed by quantifying residual beta,beta-carotene levels from extracted cell pellets by HPLC analysis (Table 1). Cells co-expressing NSC1 and NSC2 with the beta,beta-carotene pathway accumulated 60 and 40% less beta,beta-carotene compared with the empty vector control cells (Table 1). Positive control cells co-expressing AtCCD1 accumulated 84% less beta,beta-carotene, whereas cells co-expressing NSC3 accumulated similar beta,beta-carotene levels as the empty vector control cells.

However, detection of carotenoid cleavage products from culture media and organic extracts of cell pellets from bleached E. coli cultures expressing NSC1 and NSC2 proved to be difficult, because the aldehyde cleavage products appear to be further modified in E. coli. Extracts of beta,beta-carotene accumulating E. coli cells expressing NSC2 showed low levels of all-trans retinal (the 15,15'-cleavage product of beta,beta-carotene), some of which was reduced in E. coli to all-trans-retinol (supplemental Fig. S2). Carotenoid cleavage products from NSC1 co-transformed cells could not be detected. In vitro assays with whole cell lysates of NSC1-expressing cells, compared with assays with purified protein (see below), suggested that the cleavage products were nonspecifically modified by E. coli in addition to being generally not very stable. Previous studies using similar methods of co-expressing carotenoid cleavage enzymes in carotenoid-accumulating stains were also either unable to detect carotenoid cleavage products in E. coli (14) or could not account for all the cleaved beta,beta-carotene (13).

Expression of Soluble Carotenoid Cleavage Dioxygenases—The difficulties in detecting cleavage products from recombinant E. coli cells or from in vitro assays with whole cell lysates made it necessary to perform assays with purified protein. Expression of NSC enzymes using pUCmod resulted in the formation of mostly insoluble protein in E. coli. To aid in the production of soluble protein, a number of measures such as lowered growth temperatures and inducible expression were used to improve protein solubility. The genes were subcloned into the inducible expression vector pET20b+, which also provides a C-terminal histidine tag for protein purification, and induced at 18 °C overnight to slow the rate of protein formation and folding. However, despite some improvements in the expression of soluble protein, significantly improved levels of soluble proteins were only obtained when folding was aided in E. coli by the co-expression of folding chaperones. Several folding chaperones (GroEL, GroES, DnaK, DnaJ, Tf, and GrpE) provided by the Takara chaperone plasmid set were tested for their ability to promote folding of NSC enzymes in E. coli. Co-expression of NSC1, NSC2, and NSC3 with the chaperone combination GroEL and GroES produced the most soluble protein (results for NSC1 shown in Fig. 2). The soluble and C-terminally histidine-tagged proteins were purified by metal-affinity chromatography for further analysis of carotenoid cleavage. It should be noted that addition of histidine tags to the N termini of NSC proteins inactivated the enzymes, indicating a possible role of the N terminus in protein stabilization and folding or on substrate binding and/or cleavage. Unfortunately, the N terminus is not visible in the recently solved crystal structure of the 15,15'-apocarotenoid cleavage dioxygenase from Synechocystis (29) to provide an explanation for the observed inactivation.

Conditions for in Vitro Carotenoid Cleavage by NSC Enzymes—In vitro carotenoid cleavage activity of NSC enzymes was found to be strongly dependent on the solubilization of the substrates beta-apo-8'-carotenal and beta,beta-carotene. It was necessary to test a series of different assay conditions until a condition could be found under which appreciable in vitro cleavage activity of NSC enzymes was obtained. Conditions tested included different iron concentrations, reducing agents (dithiothreitol, TCEP, and ascorbate), salt concentrations (ranging from 50 to 300 mM), pH conditions, and methods of substrate solubilization. The best cleavage activity was obtained in a 50 mM phosphate buffer, pH 7.2, containing 300 mM NaCl, 0.5 mM FeSO4, 50 mM TCEP. Among all factors tested, the method of substrate solubilization had the greatest impact on the substrate cleavage rate by the enzymes. A variety of detergents and phospholipids mixed and pure micelles of lecithin as described previously (40) were tested for their ability to solubilize the hydrophobic carotenoid substrates suitable for NSC cleavage. Among several detergents tested at various concentrations (1-4% mass/volume), only carotenoid substrate solubilized with Tween 40 and beta-octylglucoside resulted in carotenoid cleavage, whereas little or no cleavage was detected with, e.g., Triton X-100, maltoglucoside, and deoxycholate. Thus, carotenoid substrates solubilized in 1% Tween 40 were used for all in vitro assays.


Figure 3
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  		FIGURE 3.
In vitro assay results from purified NSC1 dioxygenase with beta-apo-8'-carotenal as a substrate. A, HPLC chromatogram of cleavage reaction products froma2h in vitro assay with 100 µg of purified NSC1 protein and beta-apo-8'-carotenal as a substrate. Inset, UV-visible spectrum of NSC1 cleavage product. B, GC/MS chromatogram of cleavage products collected by solid phase microextraction from the headspace of a 2-h in vitro assay. The solitary peak has a retention time of 11.5 min consistent with a standard. The mass spectrum of the peak has a parent ion of 193 m/z, with the predominant fragment of 177 m/z. The retention time and mass fragmentation pattern correspond to beta-ionone.

 
With all three NSC enzymes, cleavage under in vitro conditions was only readily observed with the more soluble beta-apo-8'-carotenal. Assays with beta,beta-carotene resulted in <1% conversion of substrate into product with NSC1 and NSC2, making it difficult to analyze products. A significant lag period of 30-60 min was observed during cleavage reactions. To increase conversion rates and reduce the lag period, it was necessary to use excess protein in the assays.

All three NSC enzymes were found to require ferrous iron for activity as described for other carotenoid cleavage dioxygenases (7, 20, 21). Elemental analysis of nitric acid digests of NSC1 protein by inductively coupled plasma-mass spectrometry showed approximately a 1:1 relationship between iron metal and protein monomers after purification and reconstitution in the assay buffer, indicating that the relative slow conversion rates observed in the assays were not caused by incomplete reconstitution of the active site iron centers in the recombinant proteins.

Characterization of Carotenoid Cleavage by NSC1—To determine which double bond of the carotenoid backbone is cleaved by NSC1, beta-apo-8'-carotenal and beta,beta-carotene were used as substrates in assays with purified protein and containing iron as well as TCEP as reducing agent (7).

Cleavage of beta-apo-8'-carotenal by 100 µg of NSC1 was readily observed after 2 h by a visible shift in color of the assay solution from red to yellow (Fig. 3A). Following incubation, substrate and reaction products were extracted from the assay mixture and analyzed by HPLC. A new peak with a retention time of 10 min was detected that was not present in a control. The absorption spectrum recorded for this peak showed a {lambda}max of 422 nm and a shoulder at 444 nm, suggesting a conjugated product with more than seven double bonds (Fig. 3A). This is consistent with a C17 dialdehyde product, the result of cleavage at the 9,10 double bond of beta-apo-8'-carotenal. The molecular mass of 256 m/z confirmed the cleavage product as 8,10'-apocarotenal. The volatile second 9,10-cleavage product of beta-apo-8'-carotenal, C13 beta-ionone, was captured by solid phase micro-extraction of the headspace of the in vitro assay and identified by GC/MS using authentic beta-ionone as a standard (Fig. 3B). Cleavage of beta,beta-carotene by NSC1 after 12 h of incubation produced a very small product peak on HPLC with a slightly shorter retention time (9 min) than 8,10'-apocarotenal as would be expected for C14 dialdehyde, the 9,10,9'10'-cleavage product of beta,beta-carotene. The 9,10-cleavage product, however, was not identified (data not shown).


Figure 4
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  		FIGURE 4.
Time course of NSC1 dioxygenase cleavage of beta-apo-8'-carotenal substrate. HPLC chromatograms of cleavage reactions over four time points from in vitro assays with 100 µg of NSC1 purified protein with beta-apo-8'-carotenal as a substrate. The amount of beta-apo-8'-carotenal substrate disappears over time. Products are evident after 2 h, but after 14 h the product peak has almost completely disappeared.

 


Figure 5
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  		FIGURE 5.
In vitro assay results from purified NSC2 dioxygenase with beta-apo-8'-carotenal as a substrate. A, HPLC chromatogram of cleavage reaction products from a 2-h in vitro assay with 100-µg purified NSC2 protein and beta-apo-8'-carotenal as a substrate. The retention time of the product was 29 min, and the substrate was 48 min. B, mass spectrum of the NSC2 product. The parent mass of 285.0 m/z is consistent with a 15,15'-apocarotenal product. Inset, UV-visible spectrum of the NSC2 cleavage product with {lambda}max at 380 nm corresponding to the C15 aldehyde all-trans-retinal.

 
We noticed that, when assays of 100 µg of NSC1 with beta-apo-8'-carotenal were incubated for longer then 2-4 h, the dialdehyde cleavage product did not accumulate stoichiometrically with the decrease of substrate, suggesting breakdown of the dialdehyde product. To monitor beta-apo-8'-carotenal cleavage and accumulation of products, a time-course assay was performed with NSC1. After 0, 2, 3, 4, 5, and 14 h of incubation, aliquots of the assay were removed, extracted, and analyzed by HPLC. Fig. 4 shows that after 2 h the 9,10-cleavage product peak was apparent, but the peak did not further increase stoi-chiometrically with the decrease of the substrate peak after 5 h. After 14 h the majority of substrate was cleaved and a significant portion of the 9,10-cleavage product had degraded. Degradation products were not detected by HPLC and thought to be volatile, reactive unsaturated short chain aldehydes.

Separation of beta-apo-8'-carotenal cleavage products on the LC-MS identified a number of peaks that corresponded to geometric isomers of 8,10'-apocarotenal and additional minor cleavage products of beta-apo-8'-carotenal (supplemental Fig. S3) that are not present in a control. Spectral and mass data suggest that these additional products are the result of cleavage at the 7,8 double bond (296 m/z, {lambda}max = 442 nm) and 9',10' double bond (376 m/z, {lambda}max = 395 nm) of beta-apo-8'-carotenal (supplemental Fig. S3, A and C). This suggest that NSC1 under in vitro conditions is not absolutely specific for the 9,10 double bond.

Characterization of Carotenoid Cleavage by NSC2—Analysis of extracts from E. coli cells co-expressing NSC2 and the beta-carotene pathway identified the 15,15'-cleavage product of beta,beta-carotene, trans-retinal (supplemental Fig. S2). To confirm this result, in vitro assays were performed with purified 100 µg of NSC2 protein. After 12-16 h of incubation of 100 µg of NSC2 with either beta,beta-carotene or beta-apo-8'-carotenal a small product peak with a retention time corresponding to that of authentic trans-retinal was obtained (Fig. 5A). Absorption maximum of 380 nm and parent mass of 284 m/z of this product were consistent with trans-retinal (Fig. 5B), indicating that NSC2 is a 15,15' specific carotenoid cleavage dioxygenase. However, NSC2 was far less active in vitro than NSC1 for which beta-apo-8'-carotenal cleavage products were readily detected after 90 min of incubation.

Characterization of Carotenoid Cleavage by NSC3—The third predicted carotenoid cleavage dioxygenase in the genome of Nostoc sp. PCC 7120, NSC3, did not show in vivo cleavage activity with any of the full-length carotenoids tested. However, also included in our in vivo cleavage activity survey (Table 1) was a previously described cyanobacterial 15,15'-dioxygenase from Synechocystis PCC 6803 (SYC2) that only cleaved apocarotenoids and not full-length carotenoids (28). That report agrees with our findings that none of the full-length carotenoids tested were cleaved by this enzyme in vivo. We reasoned therefore that NSC3, like SYC2, may also be an apocarotenoid specific cleavage dioxygenase and investigated its activity in vitro with beta-apo-8'-carotenal as a substrate.


Figure 6
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  		FIGURE 6.
In vitro assay results from NSC3 dioxygenase with beta-apo-8'-carotenal as a substrate. HPLC chromatograms of cleavage reaction products from an overnight in vitro assay with 250 µg of NSC3, and 2-h in vitro assays with AtCCD1 and NSC1 with beta-apo-8'-carotenal as a substrate. The retention time of the products from NSC3, AtCCD1, and NSC1 are identical at 16 min with isomers at 17 min. Inset, UV-visible spectrum of the NSC3 cleavage product with {lambda}max at 422 nm corresponding to the C17 dialdehyde. The cleavage product is consistent with a 10,8'-apocarotendial product.

 
Cleavage of beta-apo-8'-carotenal was tested in an overnight assay with 250 µg of NSC3 purified protein at 30 °C. Samples were extracted with chloroform and subjected to LC/MS analysis to identify products. Several small product isomer peaks appeared, which all had the same molecular mass and absorption spectrum previously observed for the NSC1 C17 dialdehyde cleavage product of beta-apo-8'-carotenal (8,10'-apocarotenal) (Fig. 6). Assays with 100 µg of purified AtCCD (a 9,10,9',10' specific CCD (13)) that were extracted with chloroform resulted in an isomerized cleavage product with identical retention times and molecular mass. This confirms that NSC3 cleaves beta-apo-8'-carotenal at the 9,10 double bond.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
This study describes the isolation of three eubacterial CCD genes from Nostoc sp. PCC 7120, two of which represent microbial CCDs that cleave full-length carotenoids. From a single genome, three enzymes with three different cleavage activities were detected. NSC1 was found to cleave full-length and apocarotenoids at the 9,10 double bond, NSC2 cleaved full-length and apocarotenoids at the 15,15' double bond, and NSC3 was found to act only on apocarotenoid substrates and cleave at the 9,10 bond.

NSC1 was active against beta,beta-carotene and beta-apo-8'-carotenal in vitro with a product profile similar to AtCCD1, which is known to cleave symmetrically at the 9,10 (9'10') position. The resulting dialdehyde product from beta-apo-8'-carotenal cleavage was unstable and may explain the difficulties experienced in extracting products from co-transformation experiments with full-length carotenoids. The activity of NSC1 in vivo was less than that of AtCCD1 suggesting slow and low yielding product formation. Interestingly, time course experiments showed that there is some cleavage at alternative double bonds in addition to the 9,10 double bond. These were comparatively minor products on HPLC traces, therefore the 9,10 double bond was likely the intended target.

NSC2 cleavage products from in vivo beta,beta-carotene co-transformation experiments were detected at very low levels. The localization of the 15,15'-cleavage product to the membrane and relative stability likely prevented it from complete degradation. Although the sessile bond cleavage was similar to a previously reported cyanobacterial dioxygenase (SYC2) from Synechocystis PCC 6803, NSC2 has a broader substrate range. NSC2 converted both beta, beta-carotene and beta-apo-8'-carotenal into retinal in vitro (28). In vivo survey results also suggest that NSC2 has a stronger affinity toward linear carotenoids than NSC1 or is able to turn over faster.

NSC3 differs from its other two genomic paralogs, because it did not cleave full-length carotenoids in vivo; there was no color loss when co-expressed in carotenoid-accumulating cells. When tested in vitro with the apocarotenoid substrate, there was cleavage at the 9,10 double bond to produce 10,8'-apocarotendial. This is to our knowledge the first report of an apocarotenoid-specific dioxygenase cleaving at that double bond location. Based on reports of plant CCDs that act sequentially on carotenoids to produce small signaling molecules (12), NSC1 and NSC3 were co-expressed in an attempt to identify synergistic or sequential cleavage of carotenoids. Both in vivo and in vitro assays using beta-apo-8'-carotenal as a substrate gave no indications of new product formation.

There are several major difficulties in identifying cleavage activity and substrate specificity with the microbial CCDs. The cyanobacterial CCDs readily formed inclusion bodies making expression and purification difficult. This report identified the use of chaperone folding proteins as a potential remedy to produce more soluble protein. A more significant hurdle that still must be overcome is finding the correct conditions for substrate presentation to the enzymes. These enzymes are most likely membrane-associated, because they must access their membrane-localized carotenoid substrates. Like other enzymes, for example lipolytic enzymes, which act on hydrophobic substrates, the cleavage activity of CCDs may be strongly affected by the substrate interface in the assay. Lipases, for example, show interfacial activation at a lipid/water interface due to a conformational change in which a helical lid structure covering the active site upon contact with a hydrophobic interface moves, making the active site accessible to the hydrophobic substrate (41). It is conceivable that CCDs may likewise show interfacial activation involving conformational rearrangements. In the structure of the Synechocystis enzyme (29), loops and short helical elements surround the top part of the active site tunnel (termed "dome" region (29)) that appear to be good candidates for structural elements undergoing conformational changes similar to the lid region in lipases (42). The presence of a hydrophobic patch near the dome region suggests that this part of the protein interacts with the hydrophobic interface. The observed lag period in NSC cleavage assays with low protein concentration may point toward an interfacial activation phenomenon. The lag-burst phenomenon is typical for lipases and phospholipases. Accumulation of reaction products is assumed to trigger the burst in activity (42, 43). The use of higher NSC concentrations in the assays shortened the lag period due to faster accumulation of reaction products. Studies aimed at investigating interfacial interaction of NSC CCDs are in progress.

Hydrophobic carotenoids were difficult to solubilize and may not have been accessible for purified CCDs. The beta-apo-8'-carotenal substrate was more soluble and therefore utilized in the assays. The very low levels of activity observed for NSC1 and NSC2 against full-length carotenoids may also be the result of not identifying or testing the natural substrate of the enzymes. Endogenous carotenoids present in Nostoc PCC 7120 include beta,beta-carotene and echinenone, as well as minor carotenoids beta-cryptoxanthin, zeaxanthin, canthaxanthin, and 3'-hydroxyechinenone (44). The natural substrates for the enzymes might be unique ketocarotenoids and glycosyl carotenoids, untested apocarotenoids, or possibly not an iso-prenoid derivative. Investigations into the natural substrates of these enzymes are ongoing.

NSC1 and NSC2 could both accept linear or cyclized carotenes as well as some xanthophylls, although with different affinities. The presence of multiple dioxygenases within a single genome with different cleavage site specificities and different affinities suggest that Nostoc sp. PCC7120 CCDs may have different functions in the native host or might indicate overlapping functions or interactions between enzymes. The recent discovery of functioning rhodopsins in Nostoc sp. PCC 7120 suggests that the retinal formed by NSC2 may have a role in the photosensory signaling pathway. Retinal and its derivatives are important signaling molecules in animals, and retinal is an important chromophore in Archaea and algae. Rhodopsins in Nostoc sp. PCC 7120 are thought to act as photosensory receptors sensing green light and modulating chromatic adaptation (31).

Cleavage products other than retinal may be involved in carotenoid turnover or be involved in signaling or regulatory functions. Cleavage of full-length beta,beta-carotene by NSC1 produce beta-ionone, and beta-ionone, methylheptenone, and geranylacetone are known to inhibit the growth of various cyanobacterial species (45, 46).

In conclusion, we reported the identification of previously undescribed microbial CCDs with different cleavage activities. This is one example of a microorganism with multiple active CCDs, which cleave full-length and apocarotenoids and also have different cleavage site specificities. There are many more putative CCDs in microbial genomes still to be characterized, and their biological roles are to be determined. The multiplicity in function and wide array of carotenoid cleavage enzymes makes them attractive targets for protein engineering. Mutational studies supported by structural information and sequence alignments of the large family of CCD enzymes will provide insight into their cleavage mechanisms and substrate specificity. It will further enable design of CCDs with desired substrate and cleavage site specificities.


   FOOTNOTES
 
* This work was supported in part by National Science Foundation Grant 0332478. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S3 and Tables S1 and S2. Back

1 Supported by the NIGMS/National Institutes of Health Biotechnology Training Grant T32 GM08347. Back

2 To whom correspondence should be addressed: Dept. of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 1479 Gortner Ave., St. Paul, MN 55108. Tel.: 612-625-5782; Fax: 612-625-5780; E-mail: schmi232{at}umn.edu.

3 The abbreviations used are: CCD, carotenoid cleavage dioxygenase; NCED, nine-cis-epoxy dioxygenase; TCEP, Tris(2-carboxyethyl)phosphine hydrochloride; HPLC, high performance liquid chromatography; NSC1, -2, and -3, homologs of carotenoid cleavage dioxygenases from Nostoc sp. PCC 7120; NOP1, -2, -3, and -4, homologs of carotenoid cleavage dioxygenases from N. punctiforme; PRO1, homolog of carotenoid cleavage dioxygenases from P. marinus MIT9313; SYC1, -2, homologs of carotenoid cleavage dioxygenases from Synechocystis PCC 6803; SYO1, homolog of carotenoid cleavage dioxygenases from Synechococcus elongates PCC 7942; MmBCO, mouse beta,beta-carotene oxygenase. Back


   ACKNOWLEDGMENTS
 
We thank S. Kuslak (University of Minnesota) for providing us with mouse cDNA, Dr. P. C. Lee for constructing plasmid pAC-ispA-crtM-crtN-crtOx, Dr. R. Knurr (University of Minnesota) for elemental analysis, and Drs. R. Petri and B. Mijts for their helpful insights.



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 DISCUSSION
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