ACO
Carotenoid Oxygenase (PDB ID: 2BIW) from Synechtocystis
Created by: Gabriella Cifu

     Carotenoid Oxygenase (PDB ID: 2BIW), or specifically apocarotenoid-cleaving oxygenase (ACO), is a protein found in Synechtocystis and used to synthesize retinal, a vitamin A aldehyde, which has important roles in vision and the immune system in humans. ACO functions primarily as an enzyme, and comes from a family of oxygenases known to contain over 100 members, which affect the nutrition of numerous species of both plants and animals. Additionally, carotenoid oxygenases regulate growth and development in plants, as well as synthesize pigments in both plants and animals. The enzyme specifically crystallized and analyzed to determine the structure of carotenoid oxygenase was apocarotenoid-15,15’-oxygenase, which produces retinal in cyanobacteria (1).  

     The primary structure of ACO consists of a mixture of hydrophilic and hydrophobic residues. Residues commonly found in the primary structure include alanine, proline, leucine, isoleucine, glycine, serine, and threonine. The secondary structure of ACO consists of 38 beta sheets, nine alpha helices and random coils. The substrate, (3r)-3-Hydroxy-8’-Apocarotenol, or 3ON, attaches to ACO.  Hydrogen bonding exists between an –OH group of each subunit of the carotenoid oxygenase molecule and the substrate (1). Additionally, the Fe2+, which functions as the active site in ACO, acts as both a prosthetic group and a metal ion associated with ACO. 

     The tertiary and quaternary structures of carotenoid oxygenase consist of two associated ligands and four subunits that work together to create the active site of the enzyme. The first ligand is (3R)-3-hydroxy-8’-apocarotenol, which acts as a substrate for ACO to produce retinal. The second associated ligand is Fe (III), or FE, which functions as a dioxygen acceptor and active site for the enzyme ACO. The Fe ligands create a coordination shell that captures the oxygen in water molecules. These oxygen molecules aid in the cleavage and conversion of the substrate product molecule from cis to trans, ultimately producing 13,14-trans-retinal, and limited amounts of 13-cis-retinal. ACO contains four identical subunits, which create a 7-bladed- β-propeller with four histadines, one at the center of each subunit, and each of which holds one of the four Fe2+ active centers. The four histadines that exist in ACO are His-183, His-238, His-304, and His-484, and sit at the β-propeller axes. The histadine and Fe2+ molecules exist in an octahedral arrangement, with some locations accepting H2O and dioxygen molecules to facilitate the reaction (1). Three of the histadine molecules connect to glutamates, which take up additional positions in the octahedral arrangement. One theory, although still experimental, is that an oxygen molecule, from the dioxygen that binds to the active Fe2+ site might take the one unoccupied octahedral position (2). ACO converts the substrate, all-trans-(3R)-3-hydroxy-8’-apo-β-carotenol, to all-trans-retinal by means of the propeller structure and the resultant hydrophobic substrate entry tunnel created by the four subunits (1). Conservation of the Fe2+- ligating histadines exists amongst other members of the carotenoid oxygenase family. Above the propeller chain fold, six large loops form a rounded covering. The iron active sites can then be accessed by means of non-polar tunnels, which also serve as the attachment site for the substrate. The tunnel plays a key role in catalyzing the reaction, as the Fe2+ active site is in the first coordination shell and is therefore harder for the substrate to access. The tunnel provides a means for the substrate to bind to the active site, without the need to move through different shells to the core of each substrate (3). Numerous water molecules also exist in the first coordination shell of the iron to assist in dioxygen capture. The tendency of water molecules to be captured in the shell allows for cleavage of some of the many double bonds in carotenoid, and at select locations, to create varying products. In this case, a central cleavage to create retinal is the most common product. Without the Fe2+ ligands at the active center, this coordination shell will not be created and the mechanisms will not proceed in the same manner as no water molecules will be captured and utilized (2). 

     Additionally, a non-polar patch of leucine and phenylalanine residues exists on the surface of ACO to access non-polar substrates from the membrane of ACO (1, 3). The non-polar patch associates with non-polar patches of other coupled ACO molecules by forming micelles. The non-polar patch on the surface extracts the aforementioned substrate, and dioxygen molecules attach to the octahedral configuration of the histadines and active sites attack the 15-15’ double bond of the substrate, creating a derivative of the substrate. This cleavage results in 13’,14’-cis and 13,14-cis-aldehydes being produced, which ultimately adopt more stable trans configurations (1). Researchers have found that ACO forms only trans-apocarotenoid products (4). The structure of ACO is important, as accessibility to functional site residues dictates where oxidative cleavage and isomerization of the carotenoid may occur, which can affect the products (5). ACO was crystallized using Fe-free apocarotenoid oxygenase, which correlates with the alternative configuration of ACO discussed below. The crystallization occurs in the presence of the substrate all-trans-(3R)-3-hydroxy-8’-apo-β-carotenol, and the structure observed through X-ray diffraction. APO is the wild-type protein of carotenoid oxygenase (1).

     An alternative configuration of ACO (PDB ID: 2BIX) has two chains instead of four and two different ligands: C8E and GOL. Both are 490 residues in length and are considered monomers. A major structural difference exists as the alternative configuration is a Fe-free apo-enzyme, and thus does not have the same iron active sites as the native enzyme (6). This artificially synthesized protein allows for crystallization and analysis of the structure of carotenoid oxygenase (1).

     In a second study of the mechanism of ACO activity, researchers noted that the Fe2+ ions that function as the active site do not have a negatively charged ligand, and thus when dioxygen binds to the active site, it forms radical molecules, which ultimately form either dioxetane or epoxide intermediates. Epoxide intermediates lead to the formation of retinal aldehyde products. Through numerous models of the reaction mechanisms of ACO, researchers found that water molecules around the iron molecules in the active site dictated which of the above mechanisms and intermediates occurred. Researchers concluded that the dioxygenase mechanism, where an epoxide formed and broke, was optimal for ACO, assuming water molecules moved into the coordination shell and bonded to the irons. However, if hydrophobic side chains, such as Thr-136, existed close to the site of certain histadines, namely His-304, the water molecule was less likely to bind to the iron, and would more easily detach when dioxygen bound to the active site. When this detachment occurred, the dioxetane mechanism resulted in the more stable product. Researchers studied these mechanisms by use of a quantum chemical method that used computational modeling to evaluate the various mechanism pathways for ACO (2).

     The molecular weight of apocarotenoid oxygenase is approximately 54,286.50 Da and its isoelectric point (pI) is approximately 5.77, based on output from the ‘Compute pI/Mw tool’ of ExPASy. This tool uses a protein sequence code to calculate molecular weight and isoelectric point (7). Two databases exist to analyze the primary and tertiary structure similarities of proteins, respectively: PSI-BLAST and the Dali Server. PSI-BLAST compares the primary structure of a certain protein to the primary structures of other proteins, in order to find proteins with similar sequences. In addition, this tool may find statistical significance for the differences between primary structure matches, and can also be used to find evolutionary relationships between proteins. This search results in E-values for comparison structures, with lower E-values corresponding with a greater degree of primary structure relatedness. E- values below 0.5 indicate a high degree of similarity between query protein and protein of interest (8). The Dali Server is used to compare tertiary, or 3D, structures between proteins, again to find similarity, as well as to assess the intra-molecular distance differences between the two proteins and describe how similar the folds of the two proteins are. This server compares the query protein with proteins in the Protein Data Bank, ultimately producing a Z-score, with a higher Z-score suggesting a greater degree of tertiary structure similarity between two proteins. Z-scores above two represent a high degree of tertiary structure similarity between the query protein and the protein of interest (9). For a search of carotenoid oxygenase, the first comparison structure found to have a good degree of similarity for both of the searches is Retinal pigment epithelium-specific 65 kDa, or Rpe-65, (PDB ID: 3FSN), which was found to have an E-score of 2e-31 and a Z-score value of 39.6, suggesting a very high degree of primary structure relatedness, as well as a high degree of tertiary structure relatedness. This protein comes from Bos taurus, and has two associated ligands, FE2 and PG4. The second comparison structure from these searches is viviparous14 (VP14), a 9-cis-epoxycarotenoid dioxygenase 1 molecule (PDB ID: 3NPE), which has an E-score of 4e-28 and a Z-score of 41.8, again suggesting a very high degree of primary structure relatedness, and a higher degree of tertiary relatedness than 3FSN. This protein comes from Zea mays, commonly known as corn, and has four associated ligands: DIO, FE2, OH, and OXY. Upon quick comparison, ACO, the protein of interest, has two ligands and four side chains, while VP14, the second comparison protein, has 4 ligands and only one side chain. Also containing the conserved propeller structure of ACO, VP14 has a seven-blade β- propeller, with blades connected by antiparallel strands. The N and C terminals of the blades contribute the outermost and innermost strand, respectively. There are four α-helical components, which sit on top of the propeller.  VP14 also has a non-polar tunnel structure, allowing oxygen to reach the iron active site, although His-298 blocks the tunnel before it reaches the iron. The cleavage in VP14 focuses around the 9-cis bond and an 11-12 double bond. The hydrophobic patch on VP14 consists of residues including leucine, phenylalanine, alanine, glycine, valine and proline. VP14 supports the idea of a conserved structure existing between carotenoid oxygenase family members and further demonstrates the important biological roles of related enzymes (10). Additionally, VP14 was the first enzyme in the carotenoid oxygenase family to be identified and structurally understood, and plays a prominent role in plant growth regulation (2).