International Journal of Molecular Sciences

Review Carotenoid Cleavage Oxygenases from Microbes and Photosynthetic Organisms: Features and Functions

Oussama Ahrazem 1, Lourdes Gómez-Gómez 1, María J. Rodrigo 2, Javier Avalos 3 and María Carmen Limón 3,* 1 Instituto Botánico, Departamento de Ciencia y Tecnología Agroforestal y Genética, Facultad de Farmacia, Universidad de Castilla-La Mancha, Campus Universitario s/n, 02071 Albacete, Spain; [email protected] (O.A.); [email protected] (L.G.-G.) 2 Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC), Departamento de Ciencia de los Alimentos, Calle Catedrático Agustín Escardino 7, 46980 Paterna, Spain; [email protected] 3 Departamento de Genética, Facultad de Biología, Universidad de Sevilla, Avenida Reina Mercedes 6, 41012 Sevilla, Spain; [email protected] * Correspondence: [email protected]; Tel.: +34-954-555947

Academic Editor: Vladimír Kˇren Received: 5 September 2016; Accepted: 8 October 2016; Published: 26 October 2016 Abstract: Apocarotenoids are carotenoid-derived compounds widespread in all major taxonomic groups, where they play important roles in different physiological processes. In addition, apocarotenoids include compounds with high economic value in food and cosmetics industries. Apocarotenoid biosynthesis starts with the action of carotenoid cleavage dioxygenases (CCDs), a family of non-heme iron enzymes that catalyze the oxidative cleavage of carbon–carbon double bonds in carotenoid backbones through a similar molecular mechanism, generating aldehyde or ketone groups in the cleaving ends. From the identification of the first CCD enzyme in plants, an increasing number of CCDs have been identified in many other species, including microorganisms, proving to be a ubiquitously distributed and evolutionarily conserved enzymatic family. This review focuses on CCDs from plants, algae, fungi, and bacteria, describing recent progress in their functions and regulatory mechanisms in relation to the different roles played by the apocarotenoids in these organisms.

Keywords: algae; apocarotenoids; bacteria; carotenoid cleavage dioxygenase; fungi; plants

1. Introduction Carotenoids are a large group of terpenoid fat-soluble pigments widely distributed in nature. They are abundantly present in plants, where they are masked by the green color of chlorophyll, but they are responsible for the yellow, orange, or reddish colors of many fruits and flowers. They provide color also to many microorganisms and to some animals, including birds, fishes, and crustaceans. Chemically, the carotenoids are C40 polyene compounds with a chain of conjugated double bonds, which creates a chromophore that absorbs light in the UV and blue range of the spectrum. Carotenoids are produced by algae and plants as well as by many fungi and bacteria [1–3]. Animals are mostly unable to synthesize carotenoids, but an outstanding exception was found in certain aphids, explained by recent carotenoid biosynthetic gene horizontal transfer from fungi [4,5]. Carotenoids exert important biological functions in most living organisms, usually related with their photoprotective and light-absorbing properties. Their functions are especially relevant in plants, algae, cyanobacteria, and anoxygenic prototrophic bacteria [4,6,7], where they are indispensable in photosynthesis because of their light harvesting and protecting roles [8]. Further, carotenoids act as precursors of a vast group of bioactive compounds, the apocarotenoids, with very diverse biological functions [9].

Int. J. Mol. Sci. 2016, 17, 1781; doi:10.3390/ijms17111781 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2016, 17, 1781 2 of 38

The electron-rich polyene chain of carotenoids makes them susceptible to oxidative breakdown, leading to the generation of unspecific apocarotenoid products by random cleavage, carried out by carotenoid-unrelated enzymes such as lipoxygenases or peroxidases. However, apocarotenoids are usually the result of a biologically active process, resulting from the action of specific carotenoid cleavage dioxygenases (CCDs), frequently referred to as oxygenases (CCOs), a family of non-heme iron-type enzymes that cleave double bonds in the conjugated carbon chain of carotenoids [10]. Although the first carotenoid cleaving activity was reported in animal tissues [11], the first CCD was identified in the plant Arabidopsis thaliana [12], initiating the discovery of a large series of CCD enzymes in many other species. CCDs typically catalyze the cleavage of non-aromatic double bonds by dioxygen to form aldehyde or ketone products. Some CCDs act specifically on apocarotenoid substrates, and these enzymes are known as apocarotenoid cleavage oxygenases (ACOs). In addition to carotenogenic organisms, represented by plants, algae, fungi, and bacteria, CCDs are also widespread in animals, using them to cleave carotenoids acquired through the diet. This review covers the different CCD families identified hitherto in microorganisms and in photosynthetic species. In the microbial sections, the name CCDs will be generically used to include all types of oxygenases, and the nomenclature ACO will be reserved for the apocarotenoid specific oxygenases. In the plant section, we will refer to the CCD1, 2, 4, 7, and 8 enzyme subfamilies. The members of the nine-cis-epoxycarotenoid dioxygenases (NCED) subfamily responsible for the specific cleavage of 9-cis-epoxycarotenoids and involved in the production of abscisic acid (ABA) are not included in this review since it has been the subject of numerous studies and their activities and functions are well known [12–18].

2. Bacterial Carotenoid Oxygenases Biological roles of CCDs in bacteria are not well established. In cyanobacteria, apocarotenoids act as photoprotective and accessory pigments in thylakoid membranes. Cyanobacteria are known also to be responsible for undesired flavors in drinking water and fish from aquiculture. They produce different odor compounds such as fermentation products and apocarotenoids, the former of which is also found in microbially produced dairy food. The apocarotenoids include derivatives of β-ionone, which generally exhibit pleasant odors found in many flowers and are widely used by industry. Apocarotenoids are produced from carotenoids by CCDs, the first of which was described in Microcystis PPC 7806 [19]. A very different function is found, however, in some archaea and eubacteria, where these enzymes are essential for the biosynthesis of retinal, the chromophore for rhodopsins, or similar pumps [20–22]. In fungi, a similar function has been also described (see fungal section).

2.1. Structural Studies The first crystal structure of a CCD was determined for an apocarotenoid cleavage oxygenase (ACO) from Synechocystis sp. PCC 6803 [23]. The spatial organization resembles a propeller with seven blades, conserved in all described CCDs and, in fact, a structural signature for all of them. Five blades (I to V) are made of four antiparallel β strands, and two blades (VI and VII) consist of 5 strands (Figure1)[24]. The active center is located on the top of the enzyme, close to the propeller axis. CCDs contain a Fe2+ ion as a cofactor that is indispensable for the cleavage activity. Its putative role is to activate oxygen involved in the enzymatic reaction. The Fe2+ is coordinated by four His residues, which are conserved in the CCD family. There is a second coordination center formed by three Glu residues interacting through hydrogen bonds to three of the His residues. The requirement for these amino acids has been demonstrated via mutagenesis [25–27]. Another characteristic of CCDs is a large tunnel perpendicular to the propeller axis that enters the protein, passes through the active center, and exits the protein parallel to the propeller axis. The access to the tunnel is important for the entrance of the substrate and is located in a large hydrophobic patch that allows for the localization of the enzyme in the cell membrane. This long Int.Int. J. Mol. J. Mol. Sci. Sci.2016 2016, 17, 17, 1781, 1781 3 of3 37 of 38

patch that allows for the localization of the enzyme in the cell membrane. This long tunnel consists tunnelof hydrophobic consists of residues hydrophobic (Phe, residuesVal, Leu) (Phe,and a Val, few Leu)aromatic and aresidues few aromatic (Tyr, Trp, residues His), forming (Tyr, Trp, “van His), formingder Waals” “van forces der Waals” allowing forces a correct allowing orientation a correct of orientationthe substrate of [24]. the substrateThe hydrophobic [24]. The and hydrophobic aromatic andresidues aromatic play residues an important play an important role in roleisomerase in isomerase activity, activity, demonstrated demonstrated through through mutagenesis mutagenesis experimentsexperiments [24 [24].].

Figure 1. Cont. Int. J. Mol. Sci. 2016, 17, 1781 4 of 38 Int. J. Mol. Sci. 2016, 17, 1781 4 of 37

FigureFigure 1. 1.Tridimensional Tridimensional models models ofof 1212 carotenoid-cleavagecarotenoid-cleavage dioxygenases dioxygenases from from all all the the subfamilies subfamilies included in this review. The VP14 (PBD: 2biwA) structure from maize has been used as a template. included in this review. The VP14 (PBD: 2biwA) structure from maize has been used as a template. (A) Side view of CCDs with β-strands shown in yellow, α-helices in magenta, and loops in grey; (A) Side view of CCDs with β-strands shown in yellow, α-helices in magenta, and loops in grey; (B) Top view rotated 90° towards the viewer from (A); (C) Lateral and top views of CCD2, CCD8, and (B) Top view rotated 90◦ towards the viewer from (A); (C) Lateral and top views of CCD2, CCD8, ACO showing Fe2+ ion in green and histidines in blue. Accession numbers are: VP14: O24592.2, ACOX, and ACO showing Fe2+ ion in green and histidines in blue. Accession numbers are: VP14: O24592.2, P74334; AtCCD1, O65572; AtCCD7, AEC10494.1; AtCCD8, Q8VY26; AtCCD4: O49675; Cao-2, ACOX, P74334; AtCCD1, O65572; AtCCD7, AEC10494.1; AtCCD8, Q8VY26; AtCCD4: O49675; XP001727958.1; CarS, ADU04395.1; CarX, CAH70723.1; CsCCD2L, ALM23547.1; CcCCD4b1, Cao-2, XP001727958.1; CarS, ADU04395.1; CarX, CAH70723.1; CsCCD2L, ALM23547.1; CcCCD4b1, XP006424046; AcaA, 77754. XP006424046; AcaA, 77754. The propeller-forming β-strands are conserved between ACO (Synechocystis) and NOV2 (NovosphingobiumThe propeller-forming aromaticivoransβ-strands). However, areconserved there are structural between differences ACO (Synechocystis in the entrance) and loop NOV2and (Novosphingobiumthe dome of both aromaticivorans enzymes. The differences). However, in the there residues are structural of these structur differencesal domains in the are entrance seemingly loop andinvolved the dome in their ofboth different enzymes. substrate The specificities. differences In infact, the the residues Synechocystis of these model structural suggests differences domains are seeminglyin the substrate involved requirement in their different compared substrate with the specificities. NOV model. In In fact, ACO, the besideSynechocystiss the substratemodel tunnel, suggests differencesthere are intwo the other substrate tunnels requirement made mainly compared by hydropho with thebic NOV residues model. that In connect ACO, besides the active the substratesite to tunnel,a hydrophilic there are mouth. two other The tunnels reaction made products mainly are by di hydrophobicrected to the residuescytosol through that connect the mouth the active of the site to aexit hydrophilic tunnel. mouth. The reaction products are directed to the cytosol through the mouth of the exit tunnel. 2.2. Substrate Specificity 2.2. SubstrateStudies Specificity on bacterial CCDs usually focused the attention on the purification of different enzymes andStudies the determination on bacterial of CCDs their specificity usually focused through the th attentioneir incubation on the with purification diverse carotenoid of different substrates. enzymes andFrequently, the determination the enzymes of their exhibit specificity a high specificity, through their cleaving incubation at a certain with position diverse of carotenoid the polyene substrates. chain, Frequently,while others the are enzymes less specific. exhibit The a highavailable specificity, information cleaving is summarized at a certain below. position of the polyene chain, while others are less specific. The available information is summarized below. 2.2.1. Apocarotenoid Cleavage Oxygenases (ACO) 2.2.1. ApocarotenoidACO enzymes Cleavagecleave exclusively Oxygenases at (ACO)C15–C15′ double bonds of apo-β-carotenals. The best- knownACO ACOs enzymes are cleaveDiox1 exclusivelyfrom Synechocystis at C15–C15 sp. PCC0 double 6803 bondsand NosACO, of apo-β -carotenals.from Nostoc Thesp. PCC best-known 7120 ACOs(Table are 1). Diox1 Diox1 from cleavesSynechocystis at the C15–C15sp. PCC′ double 6803 andbond NosACO, of certain from all-transNostoc-apocarotenoids,sp. PCC 7120 such (Table as1 ). apo-β-carotenals, apo-β-carotenols, apo-8′-lycopenal, and apo-lycopenol [28,29]. The enzyme is Diox1 cleaves at the C15–C150 double bond of certain all-trans-apocarotenoids, such as apo-β-carotenals, rather unspecific, since it accepts apo-β-carotenals, apo-β-carotenols, and their 3-hydroxy derivatives, apo-β-carotenols, apo-80-lycopenal, and apo-lycopenol [28,29]. The enzyme is rather unspecific, since it with lengths ranging from C25 (12′-apo) to C35 (4′-apo), to generate a C20 retinal (or 3-hydroxy-retinal) accepts apo-β-carotenals, apo-β-carotenols, and their 3-hydroxy derivatives, with lengths ranging Int. J. Mol. Sci. 2016, 17, 1781 5 of 38 Int. J. Mol. Sci. 2016, 17, 1781 5 of 38 andInt. J. Mol.a dialdehyde Sci. 2016, 17, 1781produ ct (between C5 to C15) (Table 1). However, larger substrates, such5 of as38 βandInt.-carotene J. Mol.a dialdehyde Sci. (C 201640),, 17are, 1781 produnot cleavedct (between [29]. C5 to C15) (Table 1). However, larger substrates, such5 of as38 Int.Int.andβ-carotene J. J. Mol.NosACO,a Mol. dialdehyde Sci. Sci.(C 201640 2016),,also 17are, 1781, produ17namednot, 1781 cleavedct NSC2(between [29] [30],. Cis5 oneto Cof15 )the (Table three 1). CCDs However, of the cyanobacteriumlarger substrates, Nostoc such5 of sp. as38 5 of 38 (strainInt.βand-carotene J. Mol.NosACO,a dialdehydePCC Sci. (C 2016 7120).40 ),,also 17are, 1781 produThenamednot cleavedthreect NSC2(between enzymes [29] [30],. Cis exhibit5 oneto Cof15 different)the (Table three 1).su CCDs bstrateHowever, of preferences,the cyanobacteriumlarger substrates, as expected Nostoc such 5from of sp. as38 cooperativeand(strainInt.β-carotene J. Mol.NosACO,a dialdehydePCC Sci. (C 2016 functions7120).40 ),,also 17are, 1781 produ Thenamednot among cleavedthreect NSC2(between them enzymes [29] [30], [31]. .C is In5exhibit one tovitro, Cof15 NosACO)differentthe (Table three 1). cleavessuCCDs bstrateHowever, ofmonocyclic preferences,the cyanobacteriumlarger or substrates, acyclicas expected carotenoids Nostoc such 5from of sp. as38 andβ-carotene NosACO,a dialdehyde (C40 ),0also are produnamednot cleavedct NSC2(between [29] 0[30],. Cis5 oneto Cof15 )the (Table three 1). CCDs However, of the largercyanobacterium substrates, Nostoc such sp.as fromat(straincooperativeInt. C15J. Mol. C–PCCC15′ Sci.25 (122016 functions7120). double, -apo)17, 1781 The bonds among to three C to35 them enzymes generate(4 -apo), [31]. Inexhibit retinal. tovitro, generate NosACOdifferentIn vivo, a bleachingsucleaves Cbstrate20 retinal monocyclic preferences,activity (or of3-hydroxy-retinal) or NosACO acyclicas expected carotenoids was 5 from alsoof 38 and a dialdehyde βobservedInt.cooperativeandat(strain- carotene C15J. Mol.NosACO,a –dialdehydePCCC15′ Sci. on (C 2016 functionsβ7120). double40- ),carotene,,also 17are, 1781 produThenamed notbonds among cleavedzeathreect xanthin, NSC2 to(between them enzymesgenerate [29] [30], [31] .torulene, .C is In5exhibit retinal. one tovitro, Clycopene,of15 NosACO)differentInthe (Table vivo three and, 1).bleaching cleavessuCCDs diapocarotenedial bstrateHowever, ofmonocyclic preferences,theactivity cyanobacteriumlarger of or [ 32substrates, NosACO acyclicas,33 ]expected (Table carotenoids Nostoc wassuch 1). 5 from alsoof sp. as38 product (between C5 to C15) (Table1). However, larger substrates, such as β-carotene (C40), are not (strainatβobservedandcooperative- caroteneC15 NosACO,a –dialdehydePCCC15′ on (C functionsβ7120). double40- ),carotene,also are produThenamed not bonds among cleavedzeathreect xanthin,NSC2 to(between them enzymes generate [29] [30], [31] .torulene, .Cis Inexhibit5 retinal. one tovitro, Clycopene,of15 differentNosACO)Inthe (Table vivo three and, 1).bleaching sucleavesCCDs diapocarotenedial bstrateHowever, ofmonocyclic preferences,theactivity cyanobacteriumlarger of or [ 32substrates, NosACO acyclicas,33 expected] (Table carotenoids Nostoc wassuch 1). from also sp.as cleaved [29]. (straincooperativeandobservedβat- caroteneC15 TableNosACO,a –dialdehydePCCC15′ on 1.(C functions β7120). doubleEnzymatic40- ),carotene,also are produ Thenamed not bondsamong activity cleavedzeathreect xanthin,NSC2 to(between them enzymesof generate [29] bacterial[30], [31] .torulene, .C is Inexhibit5 retinal. one apotovitro, Clycopene,-ofcarotenoid15 NosACOdifferent)Inthe (Table vivo three and , cleavage 1).bleachingsucleaves CCDs diapocarotenedial bstrateHowever, oxygenasesofmonocyclic preferences,theactivity cyanobacteriumlarger Diox1of or [ 32substrates, NosACO acyclicas, 3and3 expected] (Table NosAcocarotenoids Nostoc wassuch 1). from also sp.as cooperativeβat(strainobserved- caroteneC15showingTableNosACO,NosACO, –PCCC15′ on 1.(C functions βproducts7120). double40Enzymatic- ),carotene,also are also The namednot bonds toamong different activitycleavedzea namedthree xanthin,NSC2 to them enzymes generateof substrates.[29] bacterialNSC2[30], [31]. torulene, . is Inexhibit retinal. one [apovitro,30 lycopene,-],ofcarotenoid NosACOdifferent isInthe onevivo three and , cleavage of bleachingcleavessu CCDs diapocarotenedialbstrate the threeoxygenasesofmonocyclic preferences,theactivity cyanobacterium CCDs Diox1of or [ 32 NosACO acyclic ofas, 3and3 expected] the (Table NosAcocarotenoids Nostoc cyanobacteriumwas 1). from also sp. Nostoc sp. atobservedcooperative(strain C15TableNosACO,showing –PCCC15′ on 1. functions βproducts7120). doubleEnzymatic- carotene,also Thenamed bonds toamong activitydifferentzeathree xanthin,NSC2 to them enzymesgenerateof substrates. bacterial[30], [31] torulene, . is Inexhibit retinal. one apovitro, lycopene,-ofcarotenoid NosACOdifferentInthe vivo three and , cleavage bleaching sucleavesCCDs diapocarotenedialbstrate oxygenasesofmonocyclic preferences,theactivity cyanobacterium Diox1of or [ 32 NosACO acyclicas, 3and3 ]expected (Table NosAcocarotenoids Nostocwas 1). from also sp. (strain PCC 7120). The three enzymes exhibitDiox1 different substrateNosAco preferences, as expected from observed(strainatcooperative C15showingTable –PCCC15′ on 1. functions βproducts7120). doubleEnzymatic-carotene,Substrates The bondsto among differentactivityzeathree xanthin, to them enzymes generateofsubstrates. bacterial [31] torulene,. Inexhibit retinal. apovitro, lycopene,-carotenoid differentNosACOIn vivo and , cleavage bleachingsucleaves diapocarotenedialbstrate oxygenasesmonocyclic preferences,activity Diox1of or [ 32 NosACO acyclicas, 3and3 ]expected (Table NosAcocarotenoids was 1). from also cooperative functions among them [31Synechocystis]. In vitroDiox1, sp. NosACO PCC 6803 cleavesNostoc monocyclicNosAco sp. PCC 7120 or acyclic carotenoids at cooperativeobservedat C15TableshowingInt.–C15′ J.on 1.Mol. functions βproducts doubleEnzymatic-carotene, Sci.Substrates 2016 bonds toamong , activitydifferent 17zea, 1781 xanthin, to them ofgenerate substrates. bacterial [31] torulene,. In retinal. apovitro, lycopene,-carotenoid NosACOIn vivo and ,cleavage bleachingcleaves diapocarotenedial oxygenasesmonocyclic activity Diox1of or [ 32 NosACOacyclic, 3and3] (Table NosAcocarotenoids was 1). also 5 of 37 0 β-apo-4′-carotenal SynechocystisDiox1 sp. PCC 6803 NostocNosAco sp. PCC 7120 C15–C15Tableshowing 1.double productsEnzymaticSubstrates bondsto differentactivity to of substrates. generate bacterial apo retinal.-carotenoidIn vivocleavage, bleaching oxygenases activity Diox1 and of NosAco NosACO was also observed atobserved C15–C15′ on β double-βcarotene,-apo- 4′ bonds-carotenal zea xanthin, to generate torulene, retinal.Synechocystis lycopene, In vivoDiox1 and, sp.bleaching diapocarotenedial PCC 6803 activity Nostoc of [ 32NosACONosAco ,sp.33 ]PCC (Table 7120was 1). also showingTable 1. productsEnzymaticSubstrates to differentactivity of substrates. bacterial apo-carotenoid cleavage oxygenases Diox1 and NosAco onobservedβ-carotene,and on a β -dialdehydeβcarotene,-apo zeaxanthin,-4′-carotenal zea xanthin,product torulene, torulene, (between lycopene,Synechocystis lycopene, C5 toRetinalDiox1 andC and sp.15 )diapocarotenedial PCC diapocarotenedial(Table 6803 1). However,Nostoc Not[32NosAco ,sp. 3detected3] [PCC 32(Tablelarger , 337120 ] 1). (Table substrates, 1). such as showingTable 1. productsEnzymaticSubstrates to differentactivity of substrates. bacterial apo-carotenoid cleavage oxygenases Diox1 and NosAco β-caroteneβ-apo (C-4′40-),carotenal are not cleaved [29].Synechocystis RetinalDiox1 sp. PCC 6803 NostocNotNosAco sp. detected PCC 7120 Tableshowing 1. productsEnzymaticSubstrates to activitydifferent of substrates. bacterial apo-carotenoidRetinal cleavage oxygenases Diox1Not detectedand NosAco TableNosACO, 1.β-apoEnzymatic-4′- carotenal also named activity NSC2 of bacterial [30],Synechocystis is apo-carotenoidoneDiox1 ofsp. thePCC three 6803 cleavage CCDsNostoc ofNosAco oxygenases sp.the PCC cyanobacterium 7120 Diox1 and Nostoc NosAco sp. showing productsβ-apoSubstrates-8′ to-carotenal different substrates. Retinal Not detected (strain PCCβ-apo -4′7120).-carotenal The three enzySynechocystismes exhibitDiox1 sp. different PCC 6803 substrateNostocNosAco sp.preferences, PCC 7120 as expected from showingβ- productsapoSubstrates-8′-carotenal to different substrates. Retinal Not detected β-apo-4′-carotenal SynechocystisRetinalDiox1 sp. PCC 6803 NostocNosAcoRetinal sp. PCC 7120 cooperativeβ-apo functions-8′-carotenal among them [31]. In vitro, NosACO cleaves monocyclic or acyclic carotenoids Substrates Retinal Not detected β-apo-4′8′-carotenal SynechocystisRetinal sp. PCC 6803 NostocRetinal sp. PCC 7120 at C15–C15Substrates′ double bonds to generate Diox1 Synechocystisretinal.Retinal In vivo,sp. PCCbleaching 6803Not activitydetected NosAco of NostocNosACOsp. PCCwas 7120also β-apo-8′4′-carotenal Retinal Retinal 0 observedββ-apo-4-apo on- 10′β-carotene,-carotenal- carotenal zeaxanthin, torulene,Retinal lycopene, and diapocarotenedialNotRetinal detected [32,33] (Table 1). β-apo-8′-carotenal β-apo-10′- carotenal RetinalRetinal Not detected Not detected Retinal Retinal β-apo-8′-carotenal Retinal Retinal Tableβ-apo 1.-10′ Enzymatic- carotenal activity of bacterial apo-carotenoid cleavage oxygenases Diox1 and NosAco β-apo-8′-carotenal Retinal Retinal β-apo-10′- carotenal Retinal Retinal showingβ products0 to different substrates. Retinal Retinal β-apo-8-apo-8′--carotenalcarotenal β-apo-10′- carotenal Retinal Retinal β-apo-12′- carotenal RetinalRetinal Retinal Retinal β-apo-10′- carotenal Retinal Diox1 Retinal NosAco Substrates β-apo-12′- carotenal Retinal Retinal β-apo-10′- carotenal Synechocystis sp. PCC 6803 Nostoc sp. PCC 7120 ββ-apo-12′0- carotenal Retinal Not detected β-apo-10-apo-10′β--carotenal carotenal-apo-4′-carotenal Retinal Retinal β-apo-12′- carotenal Retinal Not detected Retinal Retinal Retinal Not detected β-apo-10′- carotenal Retinal Retinal β-apo-12′- carotenal Retinal Not detected (3R)-3-OH-β-apo -8′-carotenal Retinal Retinal β-apo-12′- carotenal Retinal Not detected β -apo-8′-carotenal (3R)β-3-apo-12-OH-β-apo0-carotenal -8′-carotenal Retinal NotRetinal detected β-apo-12′- carotenal Retinal Retinal (3R)-3-OH-β-apo -8′-carotenal 3-OHRetinal-retinalRetinal 3RNot-3-OH detected-retinal Not detected β-apo-12′- carotenal (3R)-3-OH-β-apo -8′-carotenal 3-OH-retinal 3R-3-OH-retinal β-apo-12′β- -apo-10carotenal′-carotenal Retinal Not detected (3R)-3-OH-β-apo -8′-carotenal 3-OH-retinal 3R-3-OH-retinal (3R)-3-OH-β-apo-8 0-carotenal Retinal Retinal Not detected Retinal (3(3RR))--33--OHOH--ββ--apoapo --12′8′--carotenalcarotenal 3-OH-retinal 3R-3-OH-retinal Retinal Not detected (3(3RR))--33--OHOH--ββ--apoapo --12′8′--carotenalcarotenal 3-OH3-OH-retinal-retinal 3R-3-OH-retinal 3R-3-OH-retinal

(3R)-3-OH-β-apoβ -apo-12-12′-carotenal′-carotenal 3-OH-retinal 3R-3-OH-retinal (3R)-3-OH-β-apo -8′-carotenal 3-OH-retinal 3R-3-OH-retinal (3R)-3-OH-β-apo -12′-carotenal 3-OH-retinal Retinal 3R-3-OH-retinal Not detected (3R)-3-OHβ-β-apo -8′-0carotenal 3-OH-retinal 3R-3-OH-retinal (3(3RR)-3-OH-)-3-OH-β-apo-12-apo -12′-carotenal-carotenal 3-OH-retinal 3R-3-OH-retinal 3-OH-retinal 3R-3-OH-retinal (3R)-3-ApoOH(3-R-β8′)-3-OH--apo-lycopenal -12′-βcarotenal-apo-8 ′-carotenal 3-OH3-OH-retinal-retinal 3R-3-OH-retinal 3R-3-OH-retinal 3-OH-retinal 3R-3-OH-retinal (3R)-3-ApoOH--β8′-apo-lycopenal -12′-carotenal 3-OH-retinal 3-OH-retinal 3R-3-OH-retinal 3R-3-OH-retinal

Acycloretinal (slow) Acycloretinal (3R)-3-ApoOH--β8′0-apo-lycopenal -12′-carotenal 3-OH-retinal 3R-3-OH-retinal Apo-8Apo-8′-lycopenal-lycopenal Acycloretinal (slow) Acycloretinal (3R)-3-OH(3R-β)-3-OH--apo -12′β-carotenal-apo-12 ′ -carotenal 3-OH-retinal 3R-3-OH-retinal Apo-8′-lycopenal AcycloretinalAcycloretinal (slow) (slow) Acycloretinal Acycloretinal 3-OH-retinal 3-OH-retinal 3R-3-OH-retinal 3R-3-OH-retinal ApoApo--10′8′--lycopenallycopenal Acycloretinal (slow) Acycloretinal 3-OH-retinal 3R-3-OH-retinal Apo-10′-lycopenal Acycloretinal (slow) Acycloretinal Apo-8′0-lycopenal Apo-8′-lycopenal Apo-10Apo-10′-lycopenal-lycopenal No information Acycloretinal Apo-8′-lycopenal Acycloretinal (slow) Acycloretinal Apo-10′-lycopenal No informationNo informationAcycloretinal (slow)Acycloretinal Acycloretinal Acycloretinal Apo-8′-lycopenal Acycloretinal (slow) Acycloretinal Apo-10′-lycopenal No information Acycloretinal Acycloretinal (slow) Acycloretinal β-apo-8′-carotenol Apo-10′0-Apo-10lycopenal ′-lycopenal No information Acycloretinal Int. J. Mol. Sci. 2016β-apo-8, 17, 1781-carotenol Acycloretinal (slow) Acycloretinal 6 of 38 β-apo-8′-carotenol No informationNo information Acycloretinal Acycloretinal Apo-10′-lycopenal Int. J. Mol. Sci. 2016, 17, 1781 6 of 38 β-apo-8′-carotenol No informationRetinalRetinal AcycloretinalRetinal Retinal Apo-10′-lycopenal Int. J. Mol. Sci. 2016β, -17apo, 1781-8′-β carotenol -apo-8′-carotenol Retinal Retinal 6 of 38 Apo-10′-lycopenal No information Acycloretinal β-apo-8′-carotenol Retinal Retinal (3R)-3-OH-β-apo-8 0-carotenol No information Retinal Acycloretinal Retinal (3R)-3β-OH-apo-β-8′-apo-carotenol -8′-carotenol 3-OHRetinal-retinal 3-OHRetinal-retinal No information Acycloretinal (3R)-3-OH-β-apo -8′-carotenol 3-OHRetinal3-OH-retinal-retinal 3-OHRetinal-retinal 3-OH-retinal β-apo(3R-)-3-OH-8′-carotenol β-apo-8 ′-carotenol (3R)-3Apo-OH-8′β--apolycopenol -8′-carotenol 3-OH-retinal 3-OH-retinal β-apo-8′-carotenol Retinal Retinal (3R)-3Apo-OH--8′β-apolycopenol -8′-carotenol 3-OH-retinal 3-OH-retinal 3-OH-retinal 3-OH-retinal 0 Retinal Retinal Apo-8βApo-apo-8′-8′-lycopenol-lycopenol-carotenol No information Acycloretinal (3R)-3-OH-β-apo -8′-carotenol 3-OHRetinal-retinal 3-OHRetinal-retinal No informationNo information Acycloretinal Acycloretinal (3R)-3-OH-β-apoApo-8 -8′-carotenol′-lycopenol 3-OH-retinal 3-OH-retinal No informationRetinal No information AcycloretinalRetinal Acycloretinal (3R4)--oxo3-OH-β--apoβ-apo- 8′-carotenal8′-carotenol 3-OH-retinal 3-OH-retinal 0 (34-oxo-R)-3-OHβ-apo-8-β-apo --carotenal8′-carotenol 3-OH-retinal 3-OH-retinal 4-oxo-β-4-oxo-apo- 8′-carotenalβ-apo-8′-carotenal (3R)-3-OH-β-apo-8′-carotenol 4-oxo34-oxo-retinal-OH-retinal-retinal (slow) (slow) 3-NotOH -testedretinal Not tested 4-oxo-β-apo- 8′-carotenal 4-oxo-retinal (slow) Not tested 4-oxo-retinal (slow) Not tested

4-oxo-retinal (slow) Not tested ReferencesReferences [29][[29] [32]32 ] References [29] [32] 0 References: Enzyme : cleavesEnzyme Enzyme at thecleaves C15– atC15′ thethe double C15–C15C15–C15 [29]bond ′ ,double or equivalent bond,bond, positionsoror equivalentequivalent. [32] positions.positions. References: Enzyme cleaves at the C15–C15′ double [29]bond , or equivalent positions. [32] 2.2.2. CCDs2.2.2. withCCDs Symmetrical :with Enzyme Symmetrical cleaves Mode at ofthe ActionMode C15–C15′ of doubleAction bond , or equivalent positions. 2.2.2. CCDs with Symmetrical Mode of Action A β-carotene oxygenase from Microcystis, not identified yet, is able to carry out two symmetrical 2.2.2. CCDsA with β-carotene Symmetrical oxygenase Mode of from Action Microcystis , not identified yet, is able to carry out two symmetrical cleavagesAcleavages β-carotene on β-carotene onoxygenase β-carotene and fromzeaxanthin andMicrocystis zeaxanthin at the, not C7 identified– C8at theand C7–C8 yet,C7′– isC8′ able anddouble to C7 carry ′bonds–C8 out′ double two[34]. symmetrical It hasbonds been [34]. It has been cleavagessuggestedA β-carotene onthat β one-carotene oxygenase molecule and of fromzeaxanthin crocetindial Microcystis at and the, not twoC7 identified– moleculesC8 and C7′yet, of– isC8′β -ablecyclocitral double to carry bonds are out released two[34]. symmetrical It from has beeneach suggested that one molecule of crocetindial and two molecules of β-cyclocitral are released from each suggestedmoleculecleavages of onthat β β- caroteneone-carotene molecule [19] and. ofOn zeaxanthin crocetindial the other at hand, and the two C7one– moleculesC8 molecule and C7′ ofof– C8′β crocetindial-cyclocitral double bonds areand released two [34 ].molecules It from has beeneach of molecule of β-carotene [19]. On the other hand, one molecule of crocetindial and two molecules of moleculehydroxylsuggested -ofβ that- cyclocitralβ- caroteneone molecule would [19]. ofOn be crocetindial thereleased other from hand, and a two moleculeone molecules molecule of zeaxanthin. ofof βcrocetindial-cyclocitral areand released two molecules from each of hydroxylmoleculeCarotenoidhydroxyl- -ofβ- cyclocitralβ-carotene βoxygenases-cyclocitral would [19]. On be arewould thereleased responsible other be from hand,released a formoleculeone from moleculethe generationofa molecule zeaxanthin. of crocetindial ofof zeaxanthin.some and bacterial two molecules aromatic of compounds,hydroxylCarotenoid-β-cyclocitral as those oxygenases produced would beare by released cyanobacterialresponsible from a formolecule species the generationof zeaxanthin.the genera of Calotrix some andbacterial Plectonema aromatic. For compounds,instance,Carotenoid Plectonema as those oxygenases notatum produced PCC are by 6306 cyanobacterialresponsible and Plectonema for species the sp. generationPCCof the 7410 genera produced of Calotrix some 6 -andmethylbacterial Plectonema-5 -heptenaromatic. For-2- instance,one,compounds, β-ionone, Plectonema as 2,6,6those- notatumtrimethylcyclohexanone, produced PCC by 6306 cyanobacterial and Plectonema β-cyclocitral, species sp. PCCof 2 -thehydroxy 7410 genera produced-2,6,6 Calotrix-trimethylcyclo 6 -andmethyl Plectonema-5-hepten-hexan. For-12- one,instance, 6β--methylionone, Plectonema- 52,6,6-hepten- notatumtrimethylcyclohexanone,-2-ol, PCCdihydroactinidiolide, 6306 and Plectonema β-cyclocitral, and sp. β- iononePCC 2-hydroxy 7410-5,6 -producedepoxide.-2,6,6-trimethylcyclo Moreover,6-methyl-5 Plectonema-hepten-hexan-12- one,notatum 6β--methylionone, PCC - 563062,6,6-hepten -trimethylcyclohexanone,and-2 -Plectonemaol, dihydroactinidiolide, sp. PCC β 7410-cyclocitral, and produced β-ionone 2-hydroxy cyclogeraniol,-5,6-epoxide.-2,6,6-trimethylcyclo Moreover,4-oxo-β-ionone, Plectonema-hexan and-1- notatumdihydroone, 6-methyl -βPCC-ionol. -56306-hepten Based and -on2 -Plectonemaol, Microcystis dihydroactinidiolide, sp. data, PCC carotenoid 7410 and produced βoxygenases-ionone cyclogeraniol,-5,6 may-epoxide. be also Moreover,4 -presentoxo-β-ionone, inPlectonema biofilms and dihydrowithnotatum Phormidium -βPCC-ionol. 6306 sp.,Based and Rivularia on Plectonema Microcystis sp., and sp. data,Tolypothrix PCC carotenoid 7410 distorta produced oxygenases [35]. cyclogeraniol, may be also 4 -presentoxo-β-ionone, in biofilms and withdihydro Phormidium-β-ionol. sp.,Based Rivularia on Microcystis sp., and data,Tolypothrix carotenoid distorta oxygenases [35]. may be also present in biofilms 2.2.3.with PhormidiumCarotenoid sp.,Oxygenases Rivularia Cleavingsp., and Tolypothrix at Different distorta Positions [35 ]. 2.2.3. Carotenoid Oxygenases Cleaving at Different Positions NSC1, also known as NosCCD, is a soluble CCD from Nostoc sp. PCC 7120 that shares 44% 2.2.3. Carotenoid Oxygenases Cleaving at Different Positions homologyNSC1, with also AtCCD1 known asfrom NosCCD, Arabidopsis is a thaliana soluble, butCCD only from 26% Nostoc identity sp. with PCC NSC3, 7120 alsothat fromshares Nostoc 44% homologysp. WhenNSC1, withpurified, also AtCCD1 known NSC1 asfrom wasNosCCD, Arabidopsis incubated is a thaliana solublewith β, -butCCDapo only-8′ from-carotenal 26% Nostoc identity in sp. vitro, with PCC NSC3,HPLC 7120 alsothatchromatograms fromshares Nostoc 44% sp.showedhomology When a peak withpurified, correspondingAtCCD1 NSC1 from was Arabidopsisto incubated 8,10′-apocarotenal, thaliana with β, -butapo and only-8′ -acarotenal second26% identity product in vitro, with was NSC3,HPLC identified alsochromatograms from by GC Nostoc-MS showedassp. the When volatile a peakpurified, β corresponding-ionone NSC1 [33 was]. If to theincubated 8,10′ incubation-apocarotenal, with was β-apo kept and-8′ for-acarotenal second 5 h, the product in product vitro, was HPLC8,10′ identified-apocarotenal chromatograms by GC -wasMS asnotshowed the accumulated volatile a peak β corresponding-ionone proportionally [33]. If to the 8,10′ toincubation -apocarotenal,the disappearance was kept and fora secondof 5 h,the the productsubstrate. product was 8,10′Moreover, identified-apocarotenal bywhen GC -wasMSthe notincubationas the accumulated volatile was β -ionone prolonged proportionally [33] . toIf the 14 toincubation h, the the disappearance 8,10′ was-apocarotenal kept for of 5 h,the disappeared, the substrate. product 8,10′ andMoreover, - isomersapocarotenal when of 8,10′ wasthe- incubationapocarotenalnot accumulated was and prolonged additionalproportionally products to 14 to h, theresul the disappearanceting 8,10′ -fromapocarotenal the cleavage of the disappeared, ofsubstrate. β-apo-8′ and-Moreover,carotenal isomers at when its of C7 8,10′ –theC8- apocarotenalandincubation C9′–C10′ was andbonds, prolongedadditional were identified products to 14 h,[3 3resul the]. In ting 8,10′any -fromcase,apocarotenal the maincleavage target disappeared, of wβ-ereapo the-8′ and- carotenalC9– C10 isomers double at its of C7bond 8,10′–C8s- andapocarotenal cleavageC9′–C10′ atandbonds, other additional were positions identified products release [3 s3resul only]. In tingany minor fromcase, products thethe maincleavage that target may of w βbe-ereapo a mechanismthe-8′- C9carotenal–C10 ofdouble at eliminating its C7bond–C8s andcompeting cleavageC9′–C10′ species atbonds, other. In were positions summary, identified release in [vitro3s3 only]. In NSC1 anyminor case, cleaves products the mainat C9that target–C10 may andw beere a C9′ mechanismthe– C10′C9–C10 double ofdouble eliminating bonds bond ins competingbicyclicand cleavage carotenoids species at other. In positions andsummary, at releaseC9 –inC10 vitros only and NSC1 minor C7′ –cleavesC8′ products double at C9that – bondsC10 may and be in a C9′ mechanism monocyclic–C10′ double of carotenoids.eliminating bonds in bicyclicInterestingly,competing carotenoids species β,β.- caroteneIn andsummary, at cleavageC9 –inC10 vitro andproducts NSC1 C7′ –cleavesC8′ such double at asC9 – bondsβC10-ionone, and in C9′ monocyclicm–ethylheptenone,C10′ double carotenoids. bonds and in Interestingly,geranylacetonebicyclic carotenoids βare,β- knowncarotene and toat inhibit cleavageC9–C10 growth andproducts of C7′ some– C8′ suchcyanobacteria double as bondsβ-ionone, [35 ]. in monocyclicmethylheptenone, carotenoids. and geranylacetoneInterestingly,NSC3, also βare named,β- knowncarotene NosDiox2, to inhibitcleavage cleaves growth products β -ofapo some-8′ -carotenals suchcyanobacteria as atβ C13-ionone, [3–5C14,]. C13′methylheptenone,–C14′, and C15 –C15′and doublegeranylacetoneNSC3, bonds also in are namedvitro; known in NosDiox2, vivo to inhibit, NSC3 cleaves growth cleaved β -ofapo C some30 -compounds,8′-carotenals cyanobacteria suchat C13 [as3–5 C14,4,4′]. -d C13′iaponeurosporene–C14′, and C15 at–C15′ the doubleC13–NSC3,C14′ bonds double also in namedvitro; bond in NosDiox2, andvivo 4,4′, NSC3-diaponeurosporen cleaves cleaved β-apo C30 -compounds,8′-carotenals-4′-al at the suchat C13 C9′ as–– C10′C14,4,4′- d doubleC13′iaponeurosporene–C14′, bond. and UsingC15 at–C15′ the C40 C13doublecarotenoids–C14′ bonds double as in substrate, vitro; bond in andvivo NSC3 4,4′, NSC3 -cleaveddiaponeurosporen cleaved torulene C30 compounds, at-4′ -C15al at–C15′ the such doubleC9′ as– C10′4,4′ bond- d doubleiaponeurosporene and bond. seemingly Using at also the C40 carotenoids3,4,3′,4′C13–C14′-tetrahydrolycopene, double as substrate, bond and NSC3 but 4,4′ -cleaveddiaponeurosporenthe cleaving torulene site at-was4′ -C15al not at–C15′ theidentified doubleC9′–C10′ in bond doublethat and case; bond. seemingly however, Using also Cno40 3,4,3′,4′productscarotenoids-tetrahydrolycopene, were as detectedsubstrate, using NSC3 but lycopene cleavedthe cleaving [31]torulene. Thesite explanationatwas C15 not–C15′ identified could double be in bondthat that theand case; fully seemingly however, conjugated also no products3,4,3′,4′-tetrahydrolycopene, were detected using but lycopene the cleaving [31]. Thesite explanationwas not identified could be in that that the case; fully however, conjugated no products were detected using lycopene [31]. The explanation could be that the fully conjugated Int. J. Mol. Sci. 2016, 17, 1781 6 of 38

2.2.2. CCDs with Symmetrical Mode of Action A β-carotene oxygenase from Microcystis, not identified yet, is able to carry out two symmetrical cleavages on β-carotene and zeaxanthin at the C7–C8 and C70–C80 double bonds [34]. It has been suggested that one molecule of crocetindial and two molecules of β-cyclocitral are released from each molecule of β-carotene [19]. On the other hand, one molecule of crocetindial and two molecules of hydroxyl-β-cyclocitral would be released from a molecule of zeaxanthin. Carotenoid oxygenases are responsible for the generation of some bacterial aromatic compounds, as those produced by cyanobacterial species of the genera Calotrix and Plectonema. For instance, Plectonema notatum PCC 6306 and Plectonema sp. PCC 7410 produced 6-methyl-5-hepten-2-one, β-ionone, 2,6,6-trimethylcyclohexanone, β-cyclocitral, 2-hydroxy-2,6,6-trimethylcyclo-hexan-1-one, 6-methyl-5-hepten-2-ol, dihydroactinidiolide, and β-ionone-5,6-epoxide. Moreover, Plectonema notatum PCC 6306 and Plectonema sp. PCC 7410 produced cyclogeraniol, 4-oxo-β-ionone, and dihydro-β-ionol. Based on Microcystis data, carotenoid oxygenases may be also present in biofilms with Phormidium sp., Rivularia sp., and Tolypothrix distorta [35].

2.2.3. Carotenoid Oxygenases Cleaving at Different Positions NSC1, also known as NosCCD, is a soluble CCD from Nostoc sp. PCC 7120 that shares 44% homology with AtCCD1 from Arabidopsis thaliana, but only 26% identity with NSC3, also from Nostoc sp. When purified, NSC1 was incubated with β-apo-80-carotenal in vitro, HPLC chromatograms showed a peak corresponding to 8,100-apocarotenal, and a second product was identified by GC-MS as the volatile β-ionone [33]. If the incubation was kept for 5 h, the product 8,100-apocarotenal was not accumulated proportionally to the disappearance of the substrate. Moreover, when the incubation was prolonged to 14 h, the 8,100-apocarotenal disappeared, and isomers of 8,100-apocarotenal and additional products resulting from the cleavage of β-apo-80-carotenal at its C7–C8 and C90–C100 bonds, were identified [33]. In any case, the main target were the C9–C10 double bonds and cleavage at other positions releases only minor products that may be a mechanism of eliminating competing species. In summary, in vitro NSC1 cleaves at C9–C10 and C90–C100 double bonds in bicyclic carotenoids and at C9–C10 and C70–C80 double bonds in monocyclic carotenoids. Interestingly, β,β-carotene cleavage products such as β-ionone, methylheptenone, and geranylacetone are known to inhibit growth of some cyanobacteria [35]. NSC3, also named NosDiox2, cleaves β-apo-80-carotenals at C13–C14, C130–C140, and C15–C150 0 double bonds in vitro; in vivo, NSC3 cleaved C30 compounds, such as 4,4 -diaponeurosporene at the 0 0 0 0 0 C13–C14 double bond and 4,4 -diaponeurosporen-4 -al at the C9 –C10 double bond. Using C40 carotenoids as substrate, NSC3 cleaved torulene at C15–C150 double bond and seemingly also 3,4,30,40-tetrahydrolycopene, but the cleaving site was not identified in that case; however, no products were detected using lycopene [31]. The explanation could be that the fully conjugated double bonds of the carotenoid backbone could facilitate the entry and the binding to the active pocket of the enzyme [23]. In vitro, 4,40-diaponeurosporene and 4,40-diaponeurosporen-40-al were cleaved at more sites than in vivo. Discrepancies were observed in torulene cleavage by NSC3, which might be due to the differences among Escherichia coli strains and protein solubilisation [33]. Apocarotenals and apocarotendials were shown to have anticancer effects, suggesting that NSC3 could be used biotechnologically to produce novel bioactive compounds [31]. Several mycobacterial species are known to synthesize carotenoids; however, Mycobacterium tuberculosis does not contain carotenogenic genes, which were probably lost during evolution. Nevertheless, two ORFs coding for putative carotenoid cleavage oxygenases, Rv0654 and Rv0913c, were found in the genome of this species. Rv0654, named MtCCO from M. tuberculosis carotenoid cleavage oxygenase [36], shows a 44% similarity with the Nostoc CCO [30] and contains the four conserved His residues involved in binding the Fe2+ cofactor. Expression of MtCCO in E. coli as a GST fusion protein allowed its biochemical characterization in vitro. β-Apo-100-carotenal was mainly cleaved by this enzyme at the C13–C14 double bond and Int. J. Mol. Sci. 2016, 17, 1781 7 of 38 less frequently at the C15–C150double bond, while 3-OH-β-apo-11-carotenal was equally cleaved at both double bonds. MtCCO cleaved also C30 compounds efficiently, but exhibited only a weak activity on C25 compounds and no activity on shorter molecules, indicating that the substrate of this enzyme must have a minimal length of 25 carbons [36]. MtCCO also showed higher affinity for unsubstituted apocarotenoids, but the conversion was faster on those hydroxylated. Additionally, the enzyme cleaved 0 C40 carotenoid substrates both symmetrically at the C15–C15 and asymmetrically at the C13–C14 or C130–C140 double bonds, albeit the symmetrical and the asymmetrical cleavages were not equally targeted among the tested substrates (β-carotene, zeaxanthin, and lutein). For instance, there was a preference for a symmetrical cleavage when the β-ionone ring has a 3-OH radical (see zeaxanthin and lutein in Table2). On the other hand, the acyclic lycopene was not cleaved by MtCCO in vitro, but it was converted to apo-13-lycopenone and apo-150-lycopenal (acycloretinal) in lycopene-accumulating E. coli cells [36]. The enzyme NACOX1 from Novosphingobium aromaticivoransi exhibited cleavage activity at the C13–C14 double bound of carotenoids with a β-ionone ring giving β-apo-13-carotenone as resulting product [37]. Blast searches with the Nostoc NosCCD sequence in the genomes of the marine proteobacteria Sphingopyxis alaskensis and Plesiocystis pacifica retrieved two putative CCD genes whose identities with NosCCD ranged from 27% to 38%. After expression in carotenoid-expressing E. coli, only one of the two enzymes encoded by the putative CCD genes from both species, named respectively SaCCO and PpCCO, exhibited carotenoid cleavage activity [38]. Purified SaCCO cleaved apo-80-carotenal in vitro and released apo-120-carotenal and apo-100-carotenal (Table2); however, lycopene was poorly cleaved, and no activity was found against β-carotene or zeaxanthin. On the other hand, PpCCO cleaved zeaxanthin at the C130–C140 double bond, producing apo-130-zeaxanthinone and apo-140-zeaxanthinal, and at the C110–C120 double bond, releasing apo-120-zeaxanthinal (Table2). Similar products were observed after cantaxanthin, astaxanthin, and nostoxanthin were cleaved by PpCCO at the C130–C140 position, producing apo-130-cantaxanthinone and apo-140-cantaxanthinal (Table2), apo-13 0-astaxanthinone and apo-140-astaxanthinal (Table2), and apo-14 0-nostoxanthinal (Table3), respectively. When PpCCO cleaves cantaxanthin and nostoxanthin at their C11 0–C120 bonds, apo-120-cantaxanthinal (Table2) and apo-12 0-nostoxanthinal (Table3) are released, respectively. Int. J. Mol. Sci. 2016, 17, 1781 5 of 37

and a dialdehyde product (between C5 to C15) (Table 1). However, larger substrates, such as β-carotene (C40), are not cleaved [29]. NosACO, also named NSC2 [30], is one of the three CCDs of the cyanobacterium Nostoc sp. (strain PCC 7120). The three enzymes exhibit different substrate preferences, as expected from cooperative functions among them [31]. In vitro, NosACO cleaves monocyclic or acyclic carotenoids at C15–C15′ double bonds to generate retinal. In vivo, bleaching activity of NosACO was also observed on β-carotene, zeaxanthin, torulene, lycopene, and diapocarotenedial [32,33] (Table 1).

Table 1. Enzymatic activity of bacterial apo-carotenoid cleavage oxygenases Diox1 and NosAco showing products to different substrates.

Diox1 NosAco Substrates Synechocystis sp. PCC 6803 Nostoc sp. PCC 7120 β-apo-4′-carotenal Int. J. Mol. Sci. 2016, 17, 1781 Retinal 8 of 37 Not detected

β-apo-8′-carotenal Table 2. Enzimatic activity and substrate specificity of different bacterial carotenoid oxygenases. Retinal Retinal

Enzymes and Products β-apo-10′-carotenal Assayed Substrates β-carotene-oxygenase NosCCD (NSC1) MtCCO Mycobacterium NSC3 Nostoc sp. NACOX1 SaCCO Sphingopyxis PpCCO Plesiocystis Microcystis PCC 7806 Nostoc sp. PCC 7120 tuberculosis PCC 7120 Novosphingobium alaskensis Retinal pacifica Retinal β-ionone ( ) + Apo-8,10′ β-apo-13-carotenone Apo-12′- β-apo-8′-carotenal -apocarotene-dial ( ) β-apo-13-carotenone () ()β-apo-12 ′-carotenalβ-apo-13- carotenal ( )

Other minority products Retinal ( ) β-apo-14′-carotenal carotenone ( ) Apo-10′- Retinal Not detected ( , ) Transretinal ( ) carotenal ( )

β-apo-10′-carotenal β-ionone ( ) + Apo-10,10′ β-apo-13-carotenone () (3R)-3-OH-β-apo-8′-carotenal

-apocarotene-dial ( ) Retinal ( ) 3-OH-retinal 3R-3-OH-retinal

3-OH-β-apo-8′-carotenal 3-OH-β-ionone ( ) + Apo- 3-OH-β-apo-13-carotenone ( ) 8,10′-apocarotene 3-OH-retinal () (3R)-3-OH-β-apo-12′-carotenal -dial ( ) 3-OH-retinal 3R-3-OH-retinal 3-OH-β-apo-10′-carotenal 3-OH-β-ionone ( ) + Apo- 3-OH-β-apo-13-carotenone ( ) 10,10′-apocarotene 3-OH-retinal () -dial ( ) Apo-8′-lycopenal γ-carotene Acycloretinal (slow) Acycloretinal β-ionone ( ) + Apo-8,10′ β-apo-13-

-apocarotene-dial ( ) carotenone ( ) Apo-10′-lycopenal β-carotene β-apo-13-carotenone ( ) 2 × β-cyclocitral ( , ) Apo-10,10′-apocarotene No information Acycloretinal Retinal ( ) No No No (in vitro) No Crocetindial ( , ) -dial ( ) β-apo-14′-carotenal ( ) Int. J. Mol. Sci. 2016, 17, 1781 β-apo-8′-carotenol Apo-13′- 8 of 38 2 × hydroxi-β- 3-OH-β-apo-13-carotenone ( ) zeaxanthinone ( ) Zeaxanthin 3-OH-β-ionone ( ) Retinal Retinal cyclocitral 3-OH-β-apo-14′-carotenal ( ) Apo-14′- Apo-10,10′-apocarotene No No No (in vitro) (, ) 3-OH-retinal ( ) zeaxanthinal ( ) -dial ( ) Crocetindial ( , ) 3-OH-β-apo-11-carotenal ? (3R)-3-OH-β-apo-8′-carotenol Apo-12′- Int. J. Mol. Sci.Int. 2016 J. Mol., 17 Sci., 1781 2016 , Table17, 1781 2. EnzimaticInt. J. Mol. Sci. activity 2016, 17, 1781 and substrate specificity of different bacterial carotenoid3-OH-retinalzeaxanthinal oxygenases. ( ) 3-OH-retinal9 of 37 9 of 37 9 of 37 Lycopene Yes (in vivo) No No poorly Int. J. Mol. Sci.Int. 2016 J. Mol., 17 ,Sci. 1781 2016 , 17, 1781 Apo-8′-lycopenol Unknown8 of 37 product 9 of 37 Apo-12′- Apo-12′- Apo-12′- Int. J. Mol. Sci. 2016, 17, 1781 3-OH-β-apo-13-carotenone ( ) Enzymes and Products No information8 of 37 cantaxanthinal Acycloretinalcantaxanthinal ( ) ( ) cantaxanthinal ( ) Lutein Astaxanthin Astaxanthin 3-OH-Astaxanthin3-OH-β-iononeβ3-OH--apo-14 ( )β -ionone′-carotenal ( )( ) 3-OH-β-ionone ( ) Apo-13′- Apo-13′- Apo-13′- Int. J. Mol. Sci. 2016, 17, 1781 Table 2. Enzimatic activity and substrate specificity of different bacterial carotenoid oxygenases. 8 of Apo-1237 ′- Assayed Substrates β-carotene-oxygenase NosCCD (NSC1)3-OH, 4-oxo-Nostocβ3-OH,-inone3-OH-retinal 4-oxo- ( ) β -inone(MtCCO ) ( ) Mycobacterium 3-OH, 4-oxo-Noβ-inone NSC3 ( ) Nostoc sp. NACOX1 astaxanthinoneSaCCOastaxanthinone ( ) Sphingopyxis ( ) PpCCOastaxanthinonePlesiocystis ( ) Int. J. Mol. Sci. 2016, 17, 1781 cantaxanthinal8 of 37 ( ) Table 2. Enzimatic activity and substrateApo-10,10 specificity3-OH-′-apocaroteneApo-10,10 ofα different-apo-15′ -apocarotene′-carotenal bacterial carotenoid4-oxo-β-apo-8 oxygenases.Apo-10,10′-carotenal′-apocarotene Apo-14′- Apo-14′- Apo-14′- Int. J. Mol. Sci. 2016, 17, 1781Astaxanthin Microcystis PCC 7806 3-OH-βsp.-ionone PCC ( ) 7120 tuberculosis PCC 7120 Novosphingobium8 of Apo-1337 ′- alaskensis pacifica Table 2. Enzimatic activity and substrate specificity-dial ( of ) differentEnzymes-dial() ( )and bacterial Products carotenoid oxygenases.-dial ( ) 4-oxo-retinal (slow) astaxanthinal Notastaxanthinal ( ) tested ( ) astaxanthinal ( ) 3-OH, 4-oxo-β-inone ( ) 0 astaxanthinone ( ) Assayed Substrates β-carotene-oxygenaseReferencesTable 2. ReferencesEnzimatic NosCCDactivity (NSC1) 4-oxo-[19]and substrateβ-ionone[19] ( MtCCO specificity ) References M[30,33]ycobacterium of differentEnzymes[30,33] and bacterialNSC3 Products Nostoc [3[19]carotenoid6]sp. [3 oxygenases.6]NACOX1 [30,33][31] SaCCO Sphingopyxis[31] [37] PpCCO [36][37] Plesiocystis [38] [38][31] [38] [37] [38] [38] [38] Int. J. Mol. Sci. 20160 , 17, Echinenone1781 Apo-10,10β-ionone′-apocarotene ( ) + Apo-8,10 - β-apo-13-carotenone 8 of Apo-1437 ′- β-apo-8Assayed -carotenalSubstrates βMicrocystis-carotene-oxygenase PCC 7806 NostocNosCCD sp.Apo-10,10 PCC(NSC1) 7120′-apocarotene MtCCO tuberculosis Mycobacterium NSC3PCC Nostoc 7120 sp. NovosphingobiumNACOX1 SaCCOalaskensis Sphingopyxis PpCCOpacifica Plesiocystis 0 Symbols TableindicateSymbols 2. Enzimatic double indicateNo bonds activitydouble where andbondsapocarotene-dial substrateoxygenases-dial where Symbols( ) specificity oxygenases cleave. indicate ( of ) different Enzymes :cleave.double C7–C8; and bacterialbonds β : Products-apo-13-carotenoneC7–C8; : C9–C10; where carotenoid :oxygenases C9–C10; : ReferencesC13–C14; oxygenases. ( cleave.) : C13–C14; : C15–C15 : C7–C8; :′ ; C15–C15( ): C13 : C9–C10;′′;–C14 : ′;C13 [29] :: ′ –C14 C13–C14;astaxanthinalC11β-apo-13-′′-C12; :′ ; C11 ( ): :′ -C12C15–C15C9′–C10′; Apo-12′′:; C9 [32]′:–C10 -carotenal C13′′;–C14 ′; ( ): C11′-C12′; : C9′–C10′; Int. J. Mol. Sci. 2016, 17, 1781 Microcystis PCC 7806 β-iononeNostoc (sp. ) PCC + Apo-8,10 7120-dial (′ ) tuberculosis β-apo-13-carotenonePCC 7120 Novosphingobium alaskensisApo-12′- 0 pacifica 8 of 37 0 Int. J. Mol.Assayed Sci.β-apo-8 2016 ′-carotenalSubstrates, 17, 1781References : C7β-carotene-oxygenase′–C8′. When: C7′–C8 an′ .enzyme When[19] NosCCD an cleaves enzyme (NSC1) atOther differentcleaves[30,33] minority at: C7positions,MtCCO different′–C8 products ′M. Whenycobacterium thepositions, predominant anEnzymes enzyme[3 the6] and NSC3predominant cleavesProducts product NostocRetinal atsp. isdifferent productindicated[31] ( :) NACOX1Enzyme positions, is inindicated bold. cleaves theLackSaCCO[37] in βpredominant bold.-apo-14atof theSphingopyxisbold Lack C15–C15 means-carotenal of product bold [38] no′PpCCO double productmeans is Plesiocystis indicated bond, no carotenonepr8 product eferenceof or 37 inequivalent [38] bold. pr or (eference informationLack) positions. of or Apo-10bold information means-carotenal no product ( ) preference or information Table 2. Enzimaticβ-ionone-apocarotene-dial activity ( ) + and Apo-8,10β-ionone (substrate ) ′ ( ) β -apo-13-carotenonespecificity of different () β -apo-13-carotenonebacterial() carotenoid oxygenases.β-apo-13- carotenalApo-12 ′(- ) Assayedβ-apo-8 ′-carotenalSubstrates βMicrocystis-carotene-oxygenase PCC 7806 NostocNosCCD sp. PCC(NSC1) 7120 MtCCOtuberculosis MycobacteriumEnzymes andNSC3 ProductsPCC Nostoc 7120 sp. NovosphingobiumNACOX1 SaCCOalaskensis Sphingopyxis PpCCOpacifica Plesiocystis Int. J. Mol. Sci. 2016Symbols, 17,Canthaxantin 1781 indicatenot available. doublenot bondsavailable. where Other-apocarotene-dial oxygenases minority products (cleave. ) not ( β available.-apo-13-carotenone, : C7–C8;)Retinal ( ) : C9–C10; () β-apo-14 : ()C13–C14;′-carotenal carotenone : βC15–C15-apo-13- ( )′; : C13carotenalTransretinalApo-10′–C14 (′- ′; ) : (C11)′-C12′; 8: ofC9 37′–C10 ′; Assayed Substrates βMicrocystis-carotene-oxygenase PCC 7806 β-iononeNostocNosCCD (sp. ) PCC (NSC1)+ Apo-8,10 7120 ′ MtCCOtuberculosis Mycobacterium β-apo-13-carotenoneNSC3PCC Nostoc 7120 sp. NovosphingobiumNACOX1 SaCCOalaskensisApo-12 Sphingopyxis′- PpCCOpacifica Plesiocystis Apo-13 ′-canta- β-apo-8′-carotenal Table 2. Enzimatic activity( , and) substrate specificity of different βbacterialTransretinal-apo-14′-carotenal carotenoid ( ) oxygenases. carotenal ( ) 0 : C7′–C8′. When an enzymeTable 2. cleaves Enzimatic atOther -apocarotene-dialdifferent activity minority2 positions, xand 4-oxo-products (substrateβ )-ionone the β(predominant -apo-13-carotenonespecificity ) Retinal ( of )product differentEnzymes () 2.2.2. isβand -apo-13-carotenonebacterial indicated Products CCDs() carotenoid in with bold. Symmetrical carotenoneLack oxygenases.β-apo-13- of bold ( ) means Modecarotenal Apo-10 noof productAction ′(- ) preference or information β-apo-10 -carotenal Microcystis PCC 7806 β-iononeNostoc (sp. ) PCC+ Apo-8,10 7120 ′ tuberculosis PCC 7120 Novosphingobium alaskensisApo-12′- pacificaxanthinone ( ) Assayedββ-apo-10-apo-8′ -carotenalSubstrates′-carotenal β-carotene-oxygenase NosCCD( Apo-10,10, (NSC1)) ′-apocaroteneMtCCO Mycobacterium0 TransretinalNSC3 Nostoc (sp. ) NACOX1 SaCCOcarotenal Sphingopyxis ( ) PpCCO Plesiocystis not available. Table 2. EnzimaticβOther-ionone-apocarotene-dial activity minority ( ) + andApo-10,10β products-ionone ( substrate ) ′ ( β )-apo-13-carotenonespecificity + Apo-10,10Retinal ( of ) different - ()() β β-apo-13-carotenonebacterial-apo-14β-apo-13-carotenone()′-carotenal carotenoid carotenone oxygenases.β-apo-13-( )( ) carotenalApo-10 (′- ) Apo-14′- β-ionone ( ) + Apo-8,10′ Apo-12′- 13 ββ-apo-10-apo-8′-carotenal′-carotenal Microcystis PCC 7806 Nostoc (sp., PCC) 7120-dial ( ) tuberculosis Enzymes and ProductsPCCA β 7120-carotene Novosphingobium oxygenase from carotenalMicrocystisalaskensis ( ) , not identifiedpacifica yet, is able to carry out two symmetrical βOther-ionone-apocarotene-dial minority ( ) + Apo-10,10 products apocarotene-dial( ) ′ β-apo-13-carotenoneRetinal ( () ) Enzymes () andβTransretinal-apo-14 Products()′-carotenalRetinal ( ) ( carotenone)β-apo-13- ( ) carotenalApo-10 (′- ) cantaxanthinal ( ) Assayed Substrates β-carotene-oxygenase NosCCD (NSC1) MtCCO Mycobacterium NSC3 Nostoc sp. NACOX1 SaCCO Sphingopyxis PpCCO Plesiocystis β-apo-10′-carotenal β-ionone ((, ) +) Apo-8,10 ′ ββ-apo-13-carotenoneTransretinal-apo-14′-carotenal ( ) carotenalApo-12 (′- ) Assayedβ-apo-8 ′-carotenalSubstrates β-carotene-oxygenase Other-apocarotene-dialNosCCD minority (NSC1) products ( ) βMtCCO-apo-13-carotenoneRetinal Mycobacterium ( ) Enzymes () cleavages andNSC3 Products Nostoc onsp. β-carotenecarotenoneNACOX1 and ( ) zeaxanthinSaCCOApo-10 Sphingopyxis ′-at the C7–C8PpCCO Plesiocystis and C7′ –C8′ double bonds [34]. It has been 3-OH-β-apo-8′-carotenal Microcystis PCC 7806 3-OH-β-ionone-apocarotene-dialNostocβ-ionone ( sp. ) +PCC Apo-10,10( )7120 +( Apo- ) ′ β-apo-13-carotenonetuberculosis () PCC() 7120 Novosphingobiumβ-apo-13- carotenalalaskensis ( ) pacifica Assayedβ-apo-10 ′Substrates-carotenal0 βMicrocystis-carotene-oxygenase PCC 7806 NostocNosCCD( sp., PCC(NSC1)) 7120 3-OH-MtCCOβ-apo-13-carotenonetuberculosisInt. M ycobacteriumJ. Mol. Sci. 2016 ( ) , 17TransretinalNSC3, 1781PCC Nostoc 7120 ( sp. ) NovosphingobiumNACOX1 SaCCOcarotenalalaskensis Sphingopyxis ( ) PpCCOpacifica Plesiocystis 9 of 37 β ββ-ionone-ionone-apocarotene-dial8,10′ -apocarotene( ( ) )+ + Apo-10,10 Apo-8,10 3-OH-( ) ′ ′ β-iononeβ-apo-13-carotenoneRetinal ( () + ) () suggestedββ-apo-13-carotenone-apo-14′-carotenal that one molecule of crocetindialApo-12 ′- and two molecules of β-cyclocitral are released from each 3-OH-3-OH-β-apo-8-apo-8β-apo-8′-carotenal′-carotenal-carotenal 3-OH-Otherβ minority-ionone ( products ) + Apo- Retinal ( ) carotenone ( ) Apo-10′- β-apo-10′-carotenal Microcystis PCC 7806 β-iononeNostoc (sp. ) PCC+ Apo-8,10 7120 ′ 03-OH-3-OH-retinalβ-apo-13-carotenonetuberculosis () ( )3-OH- β-apo-13-carotenonePCCβ-apo-13-carotenone 7120 Novosphingobium ( ) alaskensisApo-12 ′- pacifica β-apo-8′-carotenal β-ionone-apocarotene-dial8,10′-dial-apocarotene (( ), +( Apo-8,10Apo-10,10) ) ( ) ′ -apocarotene-dialβ-apo-13-carotenoneRetinal ( ) () Transretinal() ( ) β-apo-13- carotenal ( ) 3-OH-β-apo-8′-carotenal 3-OH-β-ionone-apocarotene-dialβ-ionone ( ) + (Apo-8,10 ) +( Apo- ) ′ β-apo-13-carotenone3-OH-retinal () () moleculeβ-apo-13-carotenone() of β-carotene β-apo-13- [19]. On thecarotenal Apo-12other ′(- hand, ) one molecu le of crocetindial and two molecules of 3-OH-ββ-apo-10-apo-8β-apo-10′-carotenal′-carotenal′-carotenal 3-OH-Other-apocarotene-dialβ minority-ionone ( products ) +( Apo- ) 3-OH-β-apo-13-carotenoneRetinal ( ) ( ) β-apo-143-OH-retinal′-carotenal carotenone( ) ( ) Apo-10′- Apo-12′- 8,10′-dial-apocarotene ( ) 3-OH-( β)-apo-13-carotenoneRetinal ( ) ( ) β-apo-14′-carotenal 3-OH-β-apo-8′-carotenal 3-OH-βOther-ionone-apocarotene-dialβ minority-ionone (( ), + (Apo-10,10) products ) + ( Apo- ) ′ β-apo-13-carotenoneRetinal ( ) () hydroxyl-() β-cyclocitralcarotenoneβ-apo-13- would ( ) be releasedcarotenalApo-10 (′-from ) a molecule of zeaxanthin. cantaxanthinal ( ) 3-OH-β-apo-10′-carotenal 3-OH-10,10β-ionone′-apocarotene ( ) + Apo- 3-OH-3-OH-retinalβ-apo-13-carotenone () ( ) Transretinal ( ) 3-OH-β-apo-8′-carotenal 3-OH-Other-apocarotene-dial8,10β minority-ionone′-dial-apocarotene( , ( () products ) ) + ( Apo- ) 3-OH-3-OH-retinalβ-apo-13-carotenoneRetinal ( () ) ( ) βTransretinal-apo-14′-carotenal ( ) carotenone ( ) carotenalApo-10 (′- ) β-apo-10′-carotenal0 -dial ( ) 3-OH-3-OH-retinalβ-apo-13-carotenone () Astaxanthin ( ) 3-OH-β-ionone ( ) Apo-13′- 3-OH-3-OH-β-apo-10β-apo-10′-carotenal-carotenal 3-OH-β-ionone10,10β-ionone-dial′ -apocarotene(( ), (+ Apo-10,10() ) ) +3-OH- Apo- ′ β-iononeβ-apo-13-carotenone ( ) + () carotenal ( ) β-apo-10γ ′-carotenal 8,10′-apocarotene 3-OH-retinal () Transretinalβ ( ) 3-OH, 4-oxo- β-inone ( ) astaxanthinone ( ) 3-OH-β-apo-8-carotene′-carotenal 3-OH-β-iononeβ-ionone-dial ( ) (+ (Apo-10,10 ) ) + Apo-′ 03-OH-β-apo-13-carotenone3-OH-retinalβ-apo-13-carotenone () () ( )3-OH- -apo-13-carotenone ( ) 3-OH-β-apo-10β-apo-10′-carotenal′-carotenal 3-OH-β-ionone-apocarotene-dial10,10β-ionone-dial′-apocarotene ( )( +Apo-10,10 ( Apo-8,10 ) ) + ( Apo- ) ′ 3-OH--apocarotene-dialβ-apo-13-carotenoneRetinal ( ) ( ) β-apo-13- Apo-10,10 ′-apocarotene Apo-14′- γ-carotene β-ionone-apocarotene-dial8,10′-apocarotene ( ) + Apo-10,10 ( ) ′ 3-OH-β-apo-13-carotenone3-OH-retinalβ-apo-13-carotenoneRetinal ( () ) () ( ) 3-OH-retinal ( ) 3-OH-β-apo-10′-carotenal 3-OH--apocarotene-dial10,10β-ionone-dial′-apocarotene ( ( ) ) + ( Apo- ) ( 3-OH-retinal) () carotenone ( ) -dial ( ) astaxanthinal ( ) 3-OH-β-apo-8′-carotenal 3-OH-β-ionone-apocarotene-dialβ-ionone-dial ( ) (+ ( Apo-8,10 ) ) +( Apo- ) ′ 3-OH-3-OH-retinalβ-apo-13-carotenoneRetinal ( () ) ( ) β-apo-13- 39 γ-carotene -dial ( ) 3-OH-β-apo-13-carotenone ( ) 3-OH-β-apo-8′-carotenal 3-OH--apocarotene-dial10,108,10β-ionone′′-apocarotene-apocarotene ( ) + ( Apo- ) References carotenone[19] ( ) [30,33] [36] [31] [37] [38] [38] 3-OH-ββγ-apo-10-carotene′-carotenal 3-OH-β-iononeβ-ionone ( ) + ( Apo-8,10 ) + Apo-′ 3-OH-β-apo-13-carotenone3-OH-retinalβ-apo-13-carotenone () ( )( ) β-apo-13- 3-OH-γβ-carotene-apo-8-carotene′-carotenal 2 × β-cyclocitral ( , ) 3-OH-Apo-10,108,10β-ionone′-dial-apocarotene′-apocarotene ( ( ) ) + Apo- 3-OH-3-OH-retinalβ-apo-13-carotenone ()0 ( ) -apocarotene-dial10,10-dial′-apocarotene (β ) -ionone ( ) 3-OH- ( )3-OH-retinalβ +-apo-13-carotenone Apo-8,10Symbols ()- ( indicate ) double bondscarotenone where (oxygenases ) cleave. : C7–C8; : C9–C10;β-apo-13- : C13–C14; : C15–C15′; : C13′–C14′; : C11′-C12′; : C9′–C10′; βγ-carotene β-ionone-dial ( ) (+ Apo-8,10 ) ′ β-apo-13-carotenoneRetinal ( ) ( ) No β-apo-13- No No (in vitro) No 3-OH-β-apo-10-carotene′-carotenal Crocetindial ( , ) 8,10′-dial-apocarotene ( ) 3-OH-retinal () 2 × β-cyclocitral ( , ) 3-OH-βApo-10,10-ionone-apocarotene-dialβ-ionone-dial (′-apocarotene ) (+ (Apo-8,10 ) ) +( Apo- ) ′ β-apo-143-OH-retinal′-carotenal: ()C7 (′ –C8 ) ′. When an enzyme cleavescarotenoneβ-apo-13- at different ( ) positions, the predominant product is indicated in bold. Lack of bold means no product preference or information 3-OH-ββ-apo-10-carotene′-carotenal 3-OH-β-ionone-dial ( ( ) ) +apocarotene-dial Apo- 3-OH-β-apo-13-carotenoneβ-apo-13-carotenoneRetinal ( () ) ( )( ) No No No (in vitro) No carotenone ( ) γ Crocetindial ( , ) 10,10-dial′-apocarotene ( ) 3-OH-β-apo-13-carotenone ( ) 3-OH-β-apo-10-carotene′-carotenal 2 × β-cyclocitral ( , ) Apo-10,10-apocarotene-dial′-apocarotene ( ) β-apo-143-OH-retinal′-carotenal () ( ) carotenone ( ) Apo-13′- β-carotene 3-OH-β-ionone10,10β-ionone-dial′-apocarotene ( )( + ( Apo-8,10 ) ) + Apo- ′ β-apo-13-carotenoneRetinal not( ) available. ( ) No β-apo-13- No No (in vitro) No 2 ×Crocetindial β-cyclocitral2 × hydroxi- ( (β , - , ) ) Apo-10,10-dial′-apocarotene ( ) 3-OH-3-OH-retinalβ-apo-13-carotenone () ( ) zeaxanthinone ( ) βZeaxanthin 3-OH-10,10-dial′-apocaroteneβ-ionone ( ) ( ) β-apo-14′-carotenal ( ) β-apo-13-carotenoneNo No ( ) No (in vitro) Apo-13 No ′- -caroteneβγ-carotene cyclocitral -apocarotene-dial ( ) 03-OH-β-apo-13-carotenone3-OH-retinalβ-apo-14Retinal′-carotenal ( () ) ( ( ) ) carotenone ( ) Apo-14′- γ 2 Crocetindial× 2β-cyclocitral2× × hydroxi-β-cyclocitral ( (β , - , ) ) Apo-10,10 ( , -dial) ′-apocarotene (Apo-10,10 ) 3-OH--apocarotene-dialβ-apo-13-carotenone ( ) zeaxanthinone ( ) Zeaxanthin-carotene βApo-10,10-ionone3-OH- β(-ionone′-apocarotene ) + Apo-8,10 ( ) ′ β-apo-14Retinal′-carotenal ( ) ( ) No β-apo-13- No No (in vitro) Apo-13 No ′- Crocetindialcyclocitral(, () , ) β-ionone-dial ( )( + Apo-8,10 ) ′ 3-OH-3-OH-retinalβ-apo-14 ′-carotenal ( ) ( ) Retinal ( )β-apo-13- NozeaxanthinalApo-14 ′- ( ) No No (in vitro) No βγ-carotene-carotene 2 Crocetindial× hydroxi- β- ( ,-apocarotene-dial) -dial ( ) ( ) 3-OH-β(-apo-13-carotenoneβ)-apo-13-carotenone ( )( ) 0 carotenone ( ) zeaxanthinoneApo-13 ′- ( ) Zeaxanthin 2 Crocetindial× β-cyclocitral ( ( , , ) ) Apo-10,103-OH-β-ionone′-apocarotene ( ) 3-OH-β-apo-14β-apo-11-carotenal′-carotenal ( ) ? β No No No (in vitro) Apo-12′- (, ) β-ionone-apocarotene-dial ( ) + Apo-8,10 ( ) ′ 3-OH-3-OH-retinalβ-apo-14Retinal′-carotenal ( ( ) ) ( ) -apo-14No -carotenalcarotenoneβ-apo-13- No ( ) ( ) No (in vitro) zeaxanthinal No ( ) Crocetindial2 ×cyclocitral hydroxi- (β , - ) -dial ( ) 3-OH-β-apo-13-carotenone ( ) zeaxanthinoneApo-13Apo-14 ′′- ( ) Zeaxanthinβ-carotene Crocetindial ( , ) Apo-10,10-apocarotene-dial3-OH-β-ionone′-apocarotene ( ( ) ) 3-OH-β-apo-13-carotenoneβ-apo-11-carotenal ( ) ? No carotenone No ( ) No (in vitro) zeaxanthinalApo-12′- ( ) 2 × β-cyclocitralcyclocitral(, ) ( , ) Apo-10,10′-apocarotene 3-OH-3-OH-β-apo-14β3-OH-retinalβ-apo-13-carotenone-apo-14′-carotenal′-carotenal ( ( ) ( ) ( ) ) zeaxanthinalApo-14′- ( ) 0 βLycopene-carotene 2 × hydroxi-β- Apo-10,10-dial′-apocarotene ( ) β-apo-13-carotenone ( ) No No No (in vitro) zeaxanthinoneYes (in vivo) ( ) Apo-13 - Zeaxanthin 2 Crocetindial× β-cyclocitral ( ( , , ) ) Apo-10,103-OH-β-ionone′-apocarotene ( ) Retinal ( ) No No No (in vitro) zeaxanthinalApo-12Apo-13 No ′- ( ) β-carotene Crocetindialcyclocitral(, () , ) -dial ( ) 3-OH-3-OH-β-apo-13-carotenone3-OH-retinalβ-apo-14β-apo-11-carotenal′ -carotenal () ( ( ) ? ) No No Nopoorly (in vitro) zeaxanthinalApo-14 No ′- ( ) Lycopene Apo-10,10-dial′-apocarotene ( ) β-apo-14Retinal′-carotenal ( ) ( ) 3-OH-βNo-apo-13-carotenone No ( ) No (in vitro) Unknown product zeaxanthinone ( ) 2 Crocetindial× β-cyclocitral2 × hydroxi- ( (β , - , ) ) Apo-10,10-dial′-apocarotene ( ) 3-OH-3-OH-β-apo-13-carotenoneβ-apo-11-carotenal (? ) zeaxanthinonezeaxanthinalYesApo-12 (in vivo)′- ( ( ) ) ZeaxanthinZeaxanthin (,2 × )hydroxi- β-3-OH-β-ionone (3-OH- ) β-iononeβ-apo-143-OH-retinalRetinal′-carotenal ( () ( ) ( ) ) No 0 No Nopoorly (in vitro) zeaxanthinal No ( ) 0 Lycopene Crocetindialcyclocitral ( , ) -dial ( ) 3-OH-3-OH-ββ-apo-13-carotenone-apo-14′-carotenal ( ( ) ) 3-OH-β-apo-14 -carotenal ( ) UnknownzeaxanthinalApo-13Apo-14 product′- ( ) Apo-14 - Crocetindial ( , ) Apo-10,10′-apocarotene 0 3-OH-β-apo-14β-apo-11-carotenal′-carotenal ( ) ? No No No (in vitro) YesApo-12Apo-13 (in vivo)′- Lutein 2 × cyclocitral(,hydroxi- ) β- ( , ) Apo-10,103-OH-3-OH--apocarotene-dialββ-apo-13-carotenone-apo-14 ′-carotenal ( ( ) ) No No poorly Nozeaxanthinonezeaxanthinal ( ( ) ) No No (in vitro) ZeaxanthinLycopene 3-OH-β-ionone ( ) 3-OH-β3-OH-retinal-apo-13-carotenone ( ) ( ) 3-OH-retinal ( ) UnknownzeaxanthinalYes (in vivo)product ( ) zeaxanthinal ( ) 2 × hydroxi-β- -dial ( ) 3-OH-3-OH-ββ-apo-13-carotenone-apo-14′-carotenal ( ( ) ) zeaxanthinoneApo-13′- ( ) ZeaxanthinLutein CrocetindialCrocetindialcyclocitral ( , ) ( , 3-OH-) β-ionone ( ) 3-OH-3-OH-( 3-OH-retinalβ)-apo-14β-apo-11-carotenal ′-carotenal ( ) ( ? ) No NoNo poorly Apo-14Apo-12 ′- 0 Lycopene 2 ×cyclocitral hydroxi-β - Apo-10,10′-apocarotene 3-OH-3-OH-ββ-apo-13-carotenone-apo-14′-carotenal ( ( ) ) 3-OH-Noβ -apo-11-carotenal? No No (in vitro) zeaxanthinoneUnknownYesApo-14 (in vivo)product′- ( ) Apo-12 - Zeaxanthin (, ) Apo-10,103-OH-β-ionone′-apocarotene ( ) 3-OH-3-OH-retinalα-apo-15 ′-carotenal ( ) No No Nopoorly (in vitro) zeaxanthinal ( ) Lutein (, ) -dial ( ) 3-OH-3-OH-β3-OH-retinalβ-apo-13-carotenone-apo-14′-carotenal ( ) ( ( ) ) No Unknownzeaxanthinal product ( ) Crocetindialcyclocitral ( , ) 3-OH-3-OH-retinalβ-apo-11-carotenal ( ) ? Apo-12Apo-14′- Lycopene Apo-10,10-dial′-apocarotene ( ) 3-OH-α-apo-15() ′-carotenal No No No (in vitro) Yes (in vivo) zeaxanthinal ( ) Lutein Crocetindial(, () , ) 3-OH-3-OH-3-OH-β3-OH-retinalβ-apo-13-carotenone-apo-14β-apo-11-carotenal ′-carotenal ( ) ( (? ) ) No No poorly zeaxanthinalApo-12 ′- ( ) 4-oxo--dialβ-ionone ( ) ( ) Unknownzeaxanthinal product ( ) Echinenone 3-OH-α-apo-15() ′-carotenal zeaxanthinal ( ) LycopeneLycopeneLutein Crocetindial ( , ) 3-OH-3-OH-3-OH-retinalβ-apo-14β-apo-11-carotenal′-carotenal ( ) ( ? ) No Apo-12 ′- Yes (in vivo) Apo-10,104-oxo-β-ionone′-apocarotene ( ) 3-OH-β-apo-13-carotenone ( ) Yes (in vivo) EchinenoneLycopene No 3-OH-3-OH-retinalα-apo-15() ′-carotenal ( ) No No poorly NozeaxanthinalYes (in vivo) ( ) No poorly -dial ( ) No No poorly Unknown product Unknown product LycopeneLutein Apo-10,104-oxo-β-ionone′-apocarotene ( ) 3-OH-β-apo-14′-carotenal ( ) Unknown product Echinenone No 3-OH-α-apo-15() ′-carotenal Yes (in vivo) β-ionone-dial ( ( ) ) 3-OH-β3-OH-retinal-apo-13-carotenone ( ) ( ) No No poorly Apo-10,104-oxo-β-ionone′-apocarotene ( ) 3-OH-β-apo-13-carotenone() ( ) Unknown product CanthaxantinEchinenoneLutein No β-ionone ( ) 3-OH-3-OH-β-apo-14α-apo-15 ′-carotenal′-carotenal ( ) 3-OH-β-apo-13-carotenone ( ) LuteinLutein Apo-10,104-oxo--dialβ-ionone′-apocarotene ( ) ( ) 3-OH-3-OH-ββ-apo-13-carotenone-apo-14′-carotenal ( ( ) ) 0 Apo-13′-canta- CanthaxantinEchinenone No 2 x 4-oxo-β-ionone ( ) 3-OH-retinal() ( ) 3-OH-β-apo-14 -carotenalNo ( ) Lutein Apo-10,10β-ionone-dial′-apocarotene ( ( ) ) 3-OH-3-OH-retinalβ-apo-14′-carotenal ( ) ( ) No xanthinoneApo-13 ′-canta- () No Apo-10,102 x4-oxo- 4-oxo-β-iononeβ′-apocarotene-ionone ( ( ) ) 3-OH-α-apo-15 ′-carotenal No CanthaxantinEchinenone β-ionone-dial ( ( ) ) 3-OH-3-OH-retinalα-apo-15′-carotenal ( ) 3-OH-retinal ( No) xanthinoneApo-14 ′ -( ) Apo-10,10-dial′-apocarotene ( ) () 0 Apo-13′-canta- Canthaxantin No 2 x 4-oxo-β-iononeβ-ionone ( ) ( ) 3-OH-α-apo-15() ′-carotenal 3-OH-α-apo-15 -carotenal( ) cantaxanthinalApo-14 ′- ( ) 4-oxo--dialβ-ionone ( ) ( ) xanthinoneApo-13′-canta- () CanthaxantinEchinenone Apo-10,102 x4-oxo- 4-oxo-β-iononeβ′-apocarotene-ionone ( ( ) ) () cantaxanthinal ( ) Echinenone Apo-10,10β-ionone′-apocarotene ( ) xanthinoneApo-13Apo-14′-canta-′ -( ) No Apo-10,102 x 4-oxo--dialβ′-apocarotene-ionone ( ) (4-oxo- ) β-ionone ( ) Apo-10,104-oxo--dialβ-ionone′-apocarotene ( ) ( ) cantaxanthinalApo-14′- ( ) EchinenoneCanthaxantinEchinenone No -dial ( ) 0 xanthinone ( ) Apo-10,10-dial′-apocarotene (Apo-10,10 ) -apocarotene-dial Apo-13′-canta- No 2 x 4-oxo-β-iononeβ-ionone ( ) ( ) cantaxanthinalApo-14 ′- ( ) No β-ionone-dial ( ( ) ) Canthaxantin ( ) cantaxanthinalxanthinone ( ( ) ) Apo-10,10′-apocarotene Canthaxantin β-ionone ( ) Apo-13Apo-14′-canta-′- 2 x 4-oxo--dialβ-ionone ( ) ( ) β-ionone ( ) Apo-13′-canta- Canthaxantin 2 x 4-oxo-β-ionone ( ) cantaxanthinalxanthinone ( ( ) ) Apo-10,10′-apocarotene xanthinoneApo-13′-canta- () Apo-10,102 x 4-oxo-β′-apocarotene-ionone ( ) Apo-14′- -dial ( ) xanthinoneApo-14′ -( ) Apo-10,10-dial′-apocarotene ( ) cantaxanthinal ( ) cantaxanthinalApo-14′- ( ) -dial ( ) cantaxanthinal ( )

1

Int. J. Mol. Sci. 2016, 17, 1781 5 of 37

and a dialdehyde product (between C5 to C15) (Table 1). However, larger substrates, such as β-carotene (C40), are not cleaved [29]. NosACO, also named NSC2 [30], is one of the three CCDs of the cyanobacterium Nostoc sp. (strain PCC 7120). The three enzymes exhibit different substrate preferences, as expected from cooperative functions among them [31]. In vitro, NosACO cleaves monocyclic or acyclic carotenoids at C15–C15′ double bonds to generate retinal. In vivo, bleaching activity of NosACO was also observed on β-carotene, zeaxanthin, torulene, lycopene, and diapocarotenedial [32,33] (Table 1).

Table 1. Enzymatic activity of bacterial apo-carotenoid cleavage oxygenases Diox1 and NosAco showing products to different substrates.

Diox1 NosAco Substrates Int. J. Mol. Sci. 2016, 17, 1781 Synechocystis8 of 37 sp. PCC 6803 Nostoc sp. PCC 7120

Table 2. Enzimatic activity and substrate specificity of different bacterial carotenoid βoxygenases.-apo-4′-carotenal Retinal Not detected Int. J. Mol. Sci. 2016, 17, 1781 Enzymes and Products 8 of 37 Assayed Substrates β-carotene-oxygenase NosCCD (NSC1) MtCCO Mycobacterium NSC3 Nostoc sp. βNACOX1-apo-8 ′-carotenalSaCCO Sphingopyxis PpCCO Plesiocystis Microcystis PCC 7806 NostocTable sp. PCC 2. Enzimatic7120 activitytuberculosis and substrate specificityPCC 7120 of differentNovosphingobium bacterial carotenoidalaskensis oxygenases. pacifica β-ionone ( ) + Apo-8,10′ β-apo-13-carotenone Apo-12′- Retinal Retinal β-apo-8′-carotenal -apocarotene-dial ( ) β-apo-13-carotenone () () β-apo-13- carotenal ( )

Other minority products Retinal ( ) β-apo-14′-carotenalEnzymes carotenone and Products ( ) Apo-10′- Assayed Substrates β-carotene-oxygenase( , ) NosCCD (NSC1) MtCCOTransretinal Mycobacterium ( ) β-apo-10NSC3 Nostoc′-carotenalsp. carotenalNACOX1 ( ) SaCCO Sphingopyxis PpCCO Plesiocystis β-apo-10′-carotenal Microcystis PCC 7806 Nostoc sp. PCC 7120 tuberculosis PCC 7120 Novosphingobium alaskensis Retinalpacifica Retinal β-ionone ( ) + Apo-10,10′ β-iononeβ-apo-13-carotenone ( ) + Apo-8,10′ () β-apo-13-carotenone Apo-12′- β-apo-8′-carotenal -apocarotene-dial ( ) -apocarotene-dialRetinal ( ( ) ) β-apo-13-carotenone () () β-apo-13- carotenal ( )

Other minority products Retinal ( ) β-apo-14′-carotenal carotenone ( ) Apo-10′- 3-OH-β-apo-8′-carotenal 3-OH-β-ionone ( ) + Apo- 3-OH-β-apo-13-carotenone( , ) ( ) β-apo-12Transretinal′-carotenal ( ) carotenal ( ) 8,10′-apocarotene β-apo-10′-carotenal 3-OH-retinal () Retinal Not detected -dial ( ) β-ionone ( ) + Apo-10,10′ β-apo-13-carotenone ()

3-OH-β-apo-10′-carotenal 3-OH-β-ionone ( ) + Apo- 3-OH--apocarotene-dialβ-apo-13-carotenone ( ) ( ) Retinal ( ) 10,10′-apocarotene 3-OH-β-apo-8′-carotenal 3-OH-β-ionone3-OH-retinal ( ) + ()Apo- (3R)-3-OH-β-apo-8′-carotenal -dial ( ) 3-OH-β-apo-13-carotenone ( ) 8,10′-apocarotene γ-carotene 3-OH-retinal () 3-OH-retinal 3R-3-OH-retinal β-ionone ( ) + Apo-8,10′ -dial ( ) β-apo-13-

3-OH-β-apo-10′-carotenal -apocarotene-dial ( ) 3-OH-β-ionone ( ) + Apo- carotenone ( ) 3-OH-β-apo-13-carotenone ( ) 10,10′-apocarotene β-carotene β-apo-13-carotenone ( ) 3-OH-retinal () 2 × β-cyclocitral ( , ) Apo-10,10′-apocarotene -dial ( ) (3R)-3-OH-β-apo-12′-carotenal Retinal ( ) No No No (in vitro) No γCrocetindial-carotene ( , ) -dial ( ) β-iononeβ-apo-14 ( )′ -carotenal+ Apo-8,10 (′ ) β-apo-13- 3-OH-retinal 3R-3-OH-retinal

-apocarotene-dial ( ) carotenone ( ) Apo-13′- 2 × hydroxi-β- 3-OH-β-apo-13-carotenone ( ) zeaxanthinone ( ) Zeaxanthin 3-OH-β-ionone ( ) β-carotenecyclocitral 3-OH-β-apo-14′-carotenal ( ) β-apo-13-carotenone ( ) Apo-14′- 2Apo-10,10 × β-cyclocitral′-apocarotene ( , ) Apo-10,10′-apocarotene No Apo-8 No ′-lycopenal No (in vitro) (, ) 3-OH-retinal ( ) Retinal ( ) No No zeaxanthinal No (in vitro) ( ) No Int. J. Mol. Sci. 2016, 17, 1781 Crocetindial-dial ( ( ) , ) -dial ( ) 9 of 38 Crocetindial ( , ) 3-OH-β-apo-11-carotenal ? β-apo-14′-carotenal ( ) Apo-12′- Acycloretinal (slow) Acycloretinal zeaxanthinal ( ) Apo-13′- Lycopene 2 × hydroxi-β- 3-OH-β-apo-13-carotenone ( ) zeaxanthinone ( ) Zeaxanthin 3-OH-β-ionone ( ) Yes (in vivo) cyclocitral 3-OH-β-apo-14No′-carotenal ( ) Apo-10No ′-lycopenalpoorly Apo-14′- Apo-10,10′-apocarotene No No Unknown No (in product vitro) (, ) 3-OH-retinal (Int. ) J. Mol. Sci. 2016, 17, 1781 zeaxanthinal ( ) 9 of 37 3-OH-β-dial-apo-13-carotenone ( ) ( ) Table 2. Cont. No information Acycloretinal Crocetindial ( , ) 3-OH-β-apo-11-carotenal ? Apo-12′- Lutein 3-OH-β-apo-14′-carotenal ( ) zeaxanthinal ( ) 3-OH-retinal ( ) No Apo-12′- Lycopene Yes (in vivo) 3-OH-α-apo-15 ′-carotenal No No poorly cantaxanthinal ( ) β-apo-8Enzymes′-carotenol and Products Unknown product () Astaxanthin 3-OH-β-ionone ( ) Apo-13′- Assayed Substrates Int. J. Mol. Sci. 2016, 17, 1781 9 of 37 β-carotene-oxygenase4-oxo-β-iononeNosCCD ( ) (NSC1) Nostoc 3-OH-βMtCCO-apo-13-carotenoneMycobacterium ( ) NSC3 Nostoc sp. 3-OH,NACOX1 4-oxo-βRetinal-inone ( ) SaCCO Sphingopyxis PpCCORetinalPlesiocystis astaxanthinone ( ) Echinenone Lutein Apo-10,10′-apocarotene 3-OH-β-apo-14′-carotenal ( ) Apo-10,10′-apocarotene Apo-14′- MicrocystisNo PCC 7806 sp. PCC 7120 tuberculosis PCC 7120 Novosphingobium alaskensis pacifica -dial ( ) 3-OH-retinal ( ) No -dial ( ) Apo-12′- astaxanthinal ( ) 0 β-ionone ( ) 3-OH-α-apo-15′-carotenal(3R)-3-OH- Referencesβ-apo-8 ′-carotenol [19] [30,33] [36] [31]Apo-13 - [37] cantaxanthinal[38] ( ) [38] CanthaxantinCanthaxantin ()Astaxanthin 3-OH-β-ionone ( ) Apo-13′- Symbols indicate double bonds whereApo-13 oxygenases′-canta- cleave.3-OH-retinal : C7–C8;Int. J. : Mol.C9–C10; Sci. 2016 : C13–C14;, 173-OH-retinal,canta-xanthinone 1781 : C15–C15 ′; ( ): C13′–C14′; : C11′-C12′; : C9′–C10′; 9 of 37 2 x 4-oxo-β-ionone2 ( x4-oxo- ) 4-oxo-β-iononeβ-ionone (( ) 3-OH, 4-oxo-β-inone ( ) 0 astaxanthinone ( ) Echinenone : C7′–C8′. When an enzyme cleaves atxanthinone different ( positions, ) the predominant product is indicated in bold. Apo-14 Lack of bold- means no product preference or information Apo-10,10′-apocarotene Apo-10,100 ′-apocarotene Apo-10,10′-apocarotene Apo-14′- Int. J.Int. Mol. J. Mol. Sci. 2016Sci. 2016, 17, ,1781 17, 1781 No Apo-10,10 -apocarotene-dial Apo-14′ - 9 of 937 of 37 Int. J. Mol. Sci. 2016, 17, 1781 -dial ( ) -dial ( ) not available. -dial ( ) 9 of 37 cantaxanthinal ( ) astaxanthinal ( ) ( β-ionone) ( ) References Apo-8′-lycopenol[19] cantaxanthinal[30,33] ( ) [36] [31] [37] 0 [38] [38] Apo-12′- Apo-12 - Canthaxantin cantaxanthinal ( ) Symbols indicate double bonds where oxygenases cleave.Apo-12 ′ :- C7–C8;No information :Apo-13 C9–C10;′-canta- : C13–C14; Apo-12Apo-12′- ′- : C15–C15 Acycloretinal′cantaxanthinal; : C13′–C14 ′; ( ): C11′-C12′; : C9′–C10′; 2 x 4-oxo-β-ionone ( ) cantaxanthinal ( ) xanthinone ( ) cantaxanthinalAstaxanthin ( ) 3-OH-β-ionone ( ) Apo-13′- Apo-10,10′-apocarotene : C7′–C8 ′. When an enzyme cleaves at different positions, the predominant product is indicatedcantaxanthinal in bold. ( Lack ) of bold means no product0 preference or information Astaxanthin 3-OH-β-ionone (3-OH- ) β-ionone ( ) Apo-13′- Apo-14′- Apo-13 - 3-OH, 4-oxo-β-inone ( ) astaxanthinone ( ) AstaxanthinAstaxanthinAstaxanthin 3-OH-3-OH-β-iononeβ-ionone ( ) -dial( ) ( ) not available. Apo-13Apo-13′- ′- 3-OH, 4-oxo-β-inone3-OH, ( ) 4-oxo-β-inone ( ) 4-oxo-β-apo-8′-carotenal astaxanthinone ( ) cantaxanthinal ( ) astaxanthinone ( ) Apo-10,10′-apocarotene Apo-14′- Apo-10,103-OH,′-apocarotene3-OH, 4-oxo- 4-oxo-β -inone0β-inone ( ) ( ) Apo-14′- astaxanthinoneastaxanthinone ( ) ( ) 0 Apo-10,10 -apocarotene-dial 4-oxo-retinal (slow) Not testedApo-14 - -dial ( ) astaxanthinal ( ) Apo-10,10-dial Apo-10,10( ) ′-apocarotene′-apocarotene astaxanthinal ( ) Apo-14Apo-14′- ′- ( ) References astaxanthinal ( ) [19] [30,33] [36] [31] [37] [38] [38] References [19] [30,33] -dial -dial( ) ( ) [36] [31] [37] [38] [38] astaxanthinalastaxanthinal ( ) ( ) ReferencesSymbolsReferencesReferences indicate double bonds where[19] [[19]oxygenases19][ cleave. [30,33] : C7–C8;[30,33] 30 ,33 : ][C9–C10; : C13–C14;[36] [3 6] : C15–C1536][′; : ReferencesC13[31] ′–C14[31] ′; : C11′-C1231[37]][′; [37]: C9′–C10′; [38]37][ [38] [29] [38]Symbols38 [38]][ indicate [32]double 38] bonds where oxygenases cleave. : C7–C8; : C9–C10; : C13–C14; : C15–C15′; : C13′–C14′; : C11′-C12′; : C9′–C10′; SymbolsSymbols: SymbolsC7indicate′–C8 indicate′. When indicatedouble an double enzyme doublebonds cleavesbonds bondswhere at wheredifferent where oxygenases oxygenasespositions, oxygenases thecleave. predominant cleave. cleave. : C7–C8; product: C7–C8;C7–C8; is indicated : :C9–C10; C9–C10; : C9–C10; in bold. Lack: C13–C14;C13–C14; of : C13–C14;bold means : Enzyme C15–C15no : C15–C15product : C15–C15 cleaves0 pr; eference′; : C13′; :at C130or –C14the :information C13′ –C14C15–C150; ′–C14′:; C11 ′;′: 0double-C12C11: C11′0-C12; bond,′:-C12 C9′; 0–C10′ ;or : C9equivalent0:; ′–C10C9: C7′ C7–C10′;′0 –C8–C8 positions.′; ′0. . When When an an enzyme enzyme cleaves at different positions, the predominant product is indicated in bold. Lack of bold means no product preference or information not available. : C7:′ –C8C7cleaves′–C8′. When′. When at an different enzyme an enzyme positions, cleaves cleaves at the different at predominant different positions, positions, product the thepredominant isindicated predominant in product bold. product Lack is indicated is of indicated bold meansin bold. in bold. no Lack product Lack of bold of preference bold means means no or productinformationno product preference pr noteference available. or information or informationnot available. not notavailable. available. 2.2.2. CCDs with Symmetrical Mode of Action A β-carotene oxygenase from Microcystis, not identified yet, is able to carry out two symmetrical cleavages on β-carotene and zeaxanthin at the C7–C8 and C7′–C8′ double bonds [34]. It has been suggested that one molecule of crocetindial and two molecules of β-cyclocitral are released from each molecule of β-carotene [19]. On the other hand, one molecule of crocetindial and two molecules of hydroxyl-β-cyclocitral would be released from a molecule of zeaxanthin. Int. J. Mol. Sci. 2016, 17, 1781 5 of 37

and a dialdehyde product (between C5 to C15) (Table 1). However, larger substrates, such as β-carotene (C40), are not cleaved [29]. NosACO, also named NSC2 [30], is one of the three CCDs of the cyanobacterium Nostoc sp. (strain PCC 7120). The three enzymes exhibit different substrate preferences, as expected from cooperative functions among them [31]. In vitro, NosACO cleaves monocyclic or acyclic carotenoids at C15–C15′ double bonds to generate retinal. In vivo, bleaching activity of NosACO was also observed on β-carotene, zeaxanthin, torulene, lycopene, and diapocarotenedial [32,33] (Table 1).

Table 1. Enzymatic activity of bacterial apo-carotenoid cleavage oxygenases Diox1 and NosAco showing products to different substrates.

Diox1 NosAco Substrates Synechocystis sp. PCC 6803 Nostoc sp. PCC 7120 β-apo-4′-carotenal Retinal Not detected

β-apo-8′-carotenal Retinal Retinal

β-apo-10′-carotenal Retinal Retinal Int. J. Mol. Sci. 2016, 17, 1781 8 of 37

β-apo-12′-carotenal Table 2. Enzimatic activity and substrate specificity of different bacterialRetinal carotenoid oxygenases. Not detected

(3R)-3-OH-β-apo-8′-carotenal Enzymes and Products Assayed Substrates β-carotene-oxygenase NosCCD (NSC1) MtCCO Mycobacterium NSC3 Nostoc sp. NACOX1 SaCCO Sphingopyxis PpCCO Plesiocystis Microcystis PCC 7806 Nostoc sp. PCC 7120 tuberculosis PCC3-OH-retinal 7120 Novosphingobium 3R-3-OH-retinalalaskensis pacifica β-ionone ( ) + Apo-8,10′ β-apo-13-carotenone Apo-12′- β-apo-8′-carotenal -apocarotene-dial ( ) β-apo-13-carotenone () () β-apo-13- carotenal ( ) (3R)-3-OH-β-apo-12′-carotenal Other minority products Retinal ( ) β-apo-143-OH-retinal′-carotenal carotenone ( ) 3R-3-OH-retinalApo-10′- ( , ) Transretinal ( ) carotenal ( ) β-apo-10′-carotenal β-ionone ( ) + Apo-10,10′ β-apo-13-carotenone () Apo-8′-lycopenal Int. J. Mol. Sci. 2016, 17, 1781 -apocarotene-dial10 of 38 ( ) Retinal ( ) Int. J. Mol. Sci. 2016, 17, 1781 10 of 38 Acycloretinal (slow) Acycloretinal 3-OH-β-apo-8′-carotenal 3-OH-β-ionone ( ) + Apo- 3-OH-β-apo-13-carotenone ( ) 8,10′-apocarotene Apo-10′-lycopenal3-OH-retinal () -dial ( ) Table 3. Substrates tested and cleaved with a unique oxygenase. No information Acycloretinal Table 3. Substrates tested and3-OH- cleavedβ-apo-10 with′-carotenal a unique oxygenase. 3-OH-β-ionone ( ) + Apo- 3-OH-β-apo-13-carotenone ( ) Substrates Oxygenase Products 10,10′-apocarotene Int. J. Mol. Sci. 2016, 17, 1781 Substrates Oxygenase Products 3-OH-retinal10 of 38 () β-apo-4′-carotenal -dial (β ) -apo-8′-carotenol Int. J. Mol.β-apo Sci.-4′ -2016carotenal , 17, 1781 γ-carotene Retinal 9Retinal of 37 Int. J. Mol. Sci. 2016, 17, 1781 β-ionone10 (of 38 ) + Apo-8,10′ β-apo-13- Int. J. Mol. Sci. 2016, 17, 1781 NACOX1 β-apo-13- carotenone ( ) 10 of 38 NACOX1 β-apo-13-carotenone ( ) -apocarotene-dial ( ) carotenone ( ) (3R)-3-OH-β-apo-8′-carotenol Apo-12′- Table 3. Substrates tested andβ-carotene cleaved with a unique oxygenase. β-apo-13-carotenone ( ) cantaxanthinal ( ) 2 × β-cyclocitral ( , ) Apo-10,10′-apocarotene 3-OH-retinal 3-OH-retinal 3,3′-dihydroxyTable Astaxanthin3.- isorenierateneSubstrates tested and cleaved with a unique3-OH- oxygenase.β-ionone ( ) Retinal ( ) No No Apo-13′- No (in vitro) No Int. J. Mol. Sci. 2016, 173,3′, 1781-dihydroxyTable 3.- isorenierateneSubstrates tested and cleaved with a unique oxygenase.Crocetindial ( , ) 10-dial of 37 ( ) Int. J. Mol. Sci. 2016, 17, 1781 3-OH, 4-oxo-β-inone ( ) 10 of 37 β-apo-14′-carotenal ( ) astaxanthinone ( ) 3-OH-β-apo-13-carotenone ( ) Int. J. Mol. Int.Sci. J.2016 Mol., 17 Sci., 1781 2016 , 17, 1781 SubstratesSubstrates OxygenaseOxygenase Apo-10,103-OH′-apocarotene-β-apoProducts-13-carotenone Products ( ) 10 of 37 Apo-8′-lycopenol 9 of 37 Apo-14′- Apo-13′- Substrates OxygenaseMtCCO 3-OH-β-apoProducts-15′-carotenal ( ) β-apo-4′-carotenal MtCCO 3-dial-OH 2-(β ×- apohydroxi- ) -15′-carotenalβ- ( ) 3-OH-β-apo-13-carotenone ( ) No information astaxanthinal Acycloretinal ( ) zeaxanthinone ( ) β-apo-4′0-carotenal Zeaxanthin 3-OH-β-apo-14′-carotenal ( )3-OH- β-ionone ( ) Int. J. Mol. Sci. 2016, 17, β1781-apo-4Table 3.References-carotenal Substrates tested and cleaved[19] with a unique oxygenase.[30,33]cyclocitral 10[36] of 37 3-OH-[31]β-apo-14 ′-carotenal ([37] ) [38] [38] Apo-14′- 3-OH-β-apo-14′-carotenal ( Apo-10,10) ′-apocarotene Apo-12No ′- No No (in vitro) Table 3. Substrates tested and cleaved NACOX1with a unique oxygenase.β-apo(,-13- carotenone) ( ) 3-OH-retinal ( ) zeaxanthinal ( ) Int. J. Mol. Sci. 2016, 17, 1781Symbols indicate double bonds whereNACOX1NACOX1 oxygenases cleave.β-apo -13β- :carotenone -apo-13-carotenoneC7–C8; ( ) : C9–C10;10-dial of4-oxo- 37 ( ( ) ) : βC13–C14;-apo-8′-carotenal : C15–C15 ′; : C13cantaxanthinal′–C14′; (: )C11 ′-C12′; : C9′–C10′; TableSubstrates 3. Substrates tested and cleavedOxygenase with a unique oxygenase.Crocetindial Products ( , ) 3-OH-β-apo-11-carotenal ? Apo-12′- Echinenone Astaxanthin : C7EchinenoneSubstrates′–C8′. When an enzyme3-OH- β cleaves-ionone ( atOxygenase ) different positions, the predominant Products product is indicated in bold. Lack of bold means no4-oxo-retinalApo-13 product′- preference (slow) or information Not tested β-apo-4′-carotenal zeaxanthinal ( ) β-apo-4Substrates′-carotenal 3-OH, 4-oxo-β-inone (Oxygenase ) Products astaxanthinone ( ) 3,3′-dihydroxynotTable available. 3.- isorenierateneSubstrates tested and cleaLycopeneved NACOX1with a unique oxygenase.Apo β-apo-13-carotenone-10,10′-apocarotenoid ( ( ) ) Yes (in vivo) 0 3,3′-dihydroxyβ-apo-4′-carotenal-isorenieratene Apo-10,10′-apocarotene Apo-14No ′- No poorly 3,3 -dihydroxy-isorenierateneTable 3. Substrates tested and cleaved NACOX1withNosCCD a unique oxygenase.Apoβ-apo-13-carotenone-10,10′-apocarotenoid ( ( ) ) Unknown product -dial ( ) NosCCD 3-OH-2 × C13β ( -apo-13-carotenone) (References) astaxanthinal [29]( ) [32] Substrates OxygenaseNACOX1 3-OHβ-apo-13-carotenone-β-apo Products-13-13carotenone ( )( ) 3-OH-β-apo2 ×-13 C-carotenone ( ) ( ) References 3,3′-dihydroxy-isorenieratene[19] [30,33] [36] [31]0 [37] 3-OH-β-apo-13-carotenone[38] ( ) [38] 3,3′-dihydroxy-isorenierateneβ-apo-4Substrates′-carotenal OxygenaseMtCCOMtCCO 3-OH-3-OH-ββ-apo-13-carotenone3-OH--apo Products-15′-carotenalβ-apo-15 ( () -carotenal ( :) Enzyme cleaves at the C15–C15′ double bond, or equivalent positions. LuteinMtCCO 3-OH-β-apo-15′-carotenal ( ) 3-OH-β-apo-14′-carotenal ( ) β-apo-4′-carotenal MtCCO 3-OH-3-OH-ββ-apo-13-carotenone-apo-15-apo-14′′-carotenal-carotenal (( (0) ) ) Int. J. Mol. Sci. 2016, 17, 1781Symbols indicate double3,3 ′-dihydroxy-isorenieratenebonds where oxygenases cleave. : C7–C8;NACOX1 : C9–C10;3-OHβ-apo-13-carotenone- β3-OH--apo : C13–C14;-14′-βcarotenal-apo-14 ( ( ) :)-carotenal C15–C15′; ( ): C13′–C14′; : C11′-C129 ′of; 37 : C9′–C10′; Nostoxanthin MtCCO 3-OH-3-OH-β-apo-13-carotenone-apo-15 ′-carotenal ( ( ) ) 3-OH-retinal ( ) No Nostoxanthin NACOX1 3-OH-β-apo-13-carotenoneβ-apo-14′-carotenal ( ( ) ) : C7′–C8′. When an enzyme cleaves at different positions, the predominantMtCCO product 3-OH- is indicatedβ-apo-15′-carotenal in 2.2.2.bold. ( Lack )CCDs of withbold meansSymmetrical no3-OH- productα Mode-apo-15 preference′ -carotenalof Action or information 3-OH-β-apo-14′-carotenal ( ) 3,3′-dihydroxy-isorenierateneEchinenoneEchinenone Apo-14′-nostoxanthinal ( ) Apo-12′- not available. Echinenone 3-OH-3-OH-ββ-apo-13-carotenone-apo-14′-carotenal ( ( ) ) () Echinenone PpCCO Apo-14′-nostoxanthinal0 ( ) 3,3′-dihydroxy-isorenieratene PpCCO Apo-10,10Apo-12′Apo-10,10-nostoxanthinal′-apocarotenoid-apocarotenoid ( ()A )4-oxo- β-caroteneβ-ionone (( oxygenase ) fromcantaxanthinal Microcystis ( ) , not identified yet, is able to carry out two symmetrical EchinenoneMtCCO 3-OH-3-OH-Apo-ββ12′-apo-13-carotenone-apo-15-nostoxanthinal′-carotenal ( ( () ) Astaxanthin Echinenone3-OH-β -ionone ( ) NosCCDNosCCD Apo-10,10′-apocarotenoid ( ) Apo-13′- NosCCDMtCCO Apo3-OH--10,10′β-apo-142- apocarotenoid× C13′-carotenal () × ( Apo-10,10 )) ′-apocarotene 34 NosCCD Apo3-OH--10,10′β-apo-15-Noapocarotenoid ′-carotenal2cleavages ((C13) ) ( )on β-carotene and zeaxanthin at the C7–C8 and C7′–C8′ double bonds [34]. It has been 3-OH, 4-oxo-β-inone ( ) NosCCD Apo-10,102′-apocarotenoid × C13 ()( ) ( ) -dial ( ) astaxanthinone ( ) NosCCD 3-OH- β-apo-142 × C13′-carotenal ( ) ( ) HydroxylycopeneEchinenoneApo-10,10 ′-apocarotene 2 × C13 () suggestedβ-ionone that (one ) molecule of crocetindialApo-14′- and two molecules of β-cyclocitral are released from each HydroxylycopeneNostoxanthin NostoxanthinEchinenone -dial ( ) Canthaxantin Apo-10,10′-apocarotenoid ( ) astaxanthinal ( ) Nostoxanthin NosCCD Apo-14′-nostoxanthinalmolecule ( ) of β-carotene [19]. On the other hand, one molecule of crocetindial and two molecules of References [19] [30,33] [36]PpCCO [31] 0 [37] [38] [38] Apo-13′-canta- Nostoxanthin PpCCO Apo-10,10Apo-14Apo-12′Apo-14-nostoxanthinal2′Unknown-apocarotenoid × C13 () -nostoxanthinal ( ( )2 ) )x 4-oxo-β-ionone ( () ) Nostoxanthin NosCCDPpCCOPpCCO Unknown0 hydroxyl-β-cyclocitral would be released from a molecule of zeaxanthin. xanthinone ( ) Symbols indicate double bonds where oxygenases cleave. : C7–C8; : C9–C10; : C13–C14;Apo-14Apo-12 : ′′-nostoxanthinalC15–C152 × C13 () ′; (:Apo-10,10 ) C13′–C14′-apocarotene′; : C11 ′-C12′; : C9 ′–C10′; PpCCO Apo-12 -nostoxanthinal ( ) Apo-14′- HydroxylycopeneNostoxanthin ApoApo-12-14′′--nostoxanthinalnostoxanthinal ( )) -dial ( ) : C7′–C8′. When an enzyme cleaves at different positions, the predominant productPpCCO is indicated in bold.Apo-14′ Lack-nostoxanthinal of bold means ( ) no product preference or information cantaxanthinal ( ) HydroxylycopeneNostoxanthin PpCCO Apo-14Apo-12′′-nostoxanthinal-nostoxanthinal () ) 33 Dihydroxylycopene PpCCO Apo-12′-nostoxanthinalUnknown ( ) not available. HydroxylycopeneHydroxylycopene Dihydroxylycopene PpCCO Apo-14Apo-12′′-nostoxanthinal-nostoxanthinalUnknown (( ) PpCCO

PpCCOPpCCO Apo-12′-nostoxanthinalUnknown Unknown ( ) DihydroxylycopeneHydroxylycopene PpCCO Unknown DihydroxylycopeneHydroxylycopene PpCCO Unknown Hydroxylycopene Dihydroxylycopene PpCCO Unknown PpCCO Unknown Dihydroxylycopene PpCCO Unknown PpCCO Unknown Torulene DihydroxylycopeneTorulene Torulene PpCCO Unknown DihydroxylycopeneTorulene PpCCONSC3 TransretinalUnknown ( ) DihydroxylycopeneTorulene NSC3 Transretinal ( ) Dihydroxylycopene PpCCONSC3 TransretinalUnknown ( ) NSC3 Transretinal ( ) 4,4′-diapotorulene 30 4,4Torulene′-diapotoruleneTorulene PpCCONSC3 Unknown 4,4′-diapotorulene PpCCO Apo-14′-diapotorulenalUnknown ( ) 4,4′-diapotoruleneTorulene NSC3NSC3 Apo-14Transretinal′-diapotorulenalTransretinal ( ) ( ) ( ) 4,4′-diapotorulene NSC3 Apo-14Transretinal′-diapotorulenal ( ) ( ) 4,4′-diapotorulene-4-al NSC3 Apo-14′-diapotorulenal ( ) 4,44,4′-diapotorulene-4-al′-diapotorulene 4,40-diapotoruleneTorulene NSC3 Apo-10Apo-14′′-diapotorulenal-diapotorulenal ( ) 4,44,4′-diapotorulene-4-al′-diapotoruleneTorulene NSC3 Apo-10Apo-14′′-diapotorulenal-diapotorulenal (( ) 0 NSC3NSC3 Apo-10Apo-14′-diapotorulenalApo-14 -diapotorulenal ( ) ( ) 4,4′-diaponeurosporene NSC3 Transretinal ( ) 4,4′-diapotorulene-4-al NSC3 Transretinal ( ) 34 4,44,4′4,4′-diaponeurosporene′--diapotorulene-4-aldiapotorulene-4-al NSC3 Apo-14′-diaponeurosporenal ( ) 4,4′-diapotorulene-4-al NSC3 Apo-10′-diapotorulenal ( ) 04,4′-diaponeurosporene NSC3 Apo-14′-diaponeurosporenal ( ) 4,4 -diapotorulene-4-al NSC3 Apo-10′-diapotorulenal ( ) 4,4′-diapotorulene NSC3 Apo-14ApoApo-10-′10′-diaponeurosporenal′--diapotorulenaldiapotorulenal ( () ) 4,4′-diapotorulene 4,44,4′-diaponeurosporen-4′-diaponeurosporene′-al 0 4,4′-diaponeurosporen-4 ′-al NSC3 Apo-14′-diaponeurosporenalApo-10 -diapotorulenal ( ) ( ) NSC3 4,44,4′-diaponeurosporen-4′-diaponeurosporene ′-al NSC3 Apo-14Apo-′14′-diaponeurosporenal-diapotorulenal ( () ) 4,4′-diaponeurosporene NSC3 Apo-10Apo-′14′-diaponeurosporenal-diapotorulenal ( () ) 4,4′-diaponeurosporene NSC3 Apo-14Apo-10′-diaponeurosporenal ( ) NSC3 Apo-14′-diaponeurosporenal ( ) 0 4,4′-diaponeurosporen-4-diaponeurosporene ′-oic acid Apo-10′-diaponeurosporenal ( ) 334,4 ′4,4-diaponeurosporen-4′-diaponeurosporen-4 ′-oic′-al acid NSC3 Apo-14′-diaponeurosporenal ( ) 4,4′-diapotorulene-4-al NSC3 Apo-10ApoApo-14-14′′′-diaponeurosporenal-diaponeurosporenal0 ( ) ) 4,4′4,4-diaponeurosporen-44,4′′-diaponeurosporen-4-diapotorulene-4′-oic-al′ -al acid NSC3NSC3 Apo-14 -diaponeurosporenal ( ) NSC3 Apo-14Apo-10′′-diaponeurosporenal-diaponeurosporenal (( ) NSC3 Apo-10′-diaponeurosporenal ( ) NSC3 Apo-10Apo-′10′-diaponeurosporenal-diapotorulenal ( () ) 4,4′4,4′-diaponeurosporen-4-diaponeurosporenMyxol ′-oic-4′-al acid NSC3 Apo-10′-diapotorulenal ( ) 4,4′-diaponeurosporenMyxol -4′-al 3-OH-β-ionone ( ) 4,40 ′-diaponeurosporen-4 ′-oic acid0 4,4 -diaponeurosporen-4Myxol -al NosCCDNSC3 Apo-103-OH-′-diaponeurosporenalβ-ionone ( ) ( ) NosCCD ApoApo-8-14′′-10-apocarotenedial-diaponeurosporenal0 ( ( ) ) NSC3 ApoApo-10Apo-8-14′3-OH-Apo-14′′-10-apocarotenedial--diaponeurosporenaldiaponeurosporenalβ-ionone-diaponeurosporenal ( ) ( ( ) ) ( ) 4,4′-diaponeurosporene NosCCDNSC3NSC3 Apo-10′-diaponeurosporenal ( ) 4,4′-diaponeurosporene ApoApo-8-10′′-10-apocarotenedial-diaponeurosporenal0 ( ( ) ) Myxol-2Myxol′-fucoside Apo-10 -diaponeurosporenal ( )

Myxol-2Myxol′-fucoside 3-OH-β-ionone ( ) 34 NosCCDNSC3 Apo-14′3-OH--diaponeurosporenalβ-ionone ( ) ( ) 0 4,4′-diaponeurosporenMyxol-2′-fucoside-4′- oic0 acid NSC3 ApoApo-8-14′3-OH-′--10-apocarotenedialdiaponeurosporenalβ-ionone ( ) ( ( ) ) 4,4′-diaponeurosporen-4′-oic acid NosCCD Apo-83-OH-′-10-apocarotenedialβ-ionone ( ) ( ) 4,4 -diaponeurosporen-4 -oic acid NosCCD NosCCD Apo-83-OH-′-10-apocarotenedialβ-ionone ( ) ( ) 30 acetate adduct0 C10 Myxol-2′ -fucoside NosCCDNSC3NSC3 ApoApo-8-10′Apo-10acetate′-10-apocarotenedial-diaponeurosporenal adduct-diaponeurosporenal C10 ( ( ) ) ( ) 4,4′-diaponeurosporen-4′-al NSC3 Apo-10′-diaponeurosporenal ( ) 4,4′-diaponeurosporenMyxol-2′-fucoside- 4′-al 3-OH-acetateβ -iononeadduct C10( ) Int. J. Mol. Sci. 2016, 17, 1781 11 of 38 4-ketomyxol-2 ′-fucoside 13 4-ketomyxol-2 ′-fucoside NosCCD ApoApo-8-14′3-OH-′-10-apocarotenedial-diaponeurosporenalβ-ionone ( ) ( ( ) ) NSC3 Apo-14′-diaponeurosporenal ( ) 13 NosCCDNSC3 ApoApo-8-10′′acetate-10-apocarotenedial-diaponeurosporenal adduct C10 ( ( ) ) 4-ketomyxol-2Myxol ′-fucoside Apo-10′3-diaponeurosporenalOH-β-ionone ( ) ( ) 33 Myxol NosCCD 3-OH-4-oxo-3acetate-OH-β -adductiononeβ-ionone C10( () ) Myxol NosCCD Apo3-OH-4-oxo--8′-10-apocarotenedialβ3-OH--ionone (β -ionone( ) ) ( ) NosCCDNosCCD Apo-8Apo-8′′-10-apocarotenedial-10-apocarotenedial ( ) 4-ketomyxol-2 ′-fucoside Apo-83-OH-4-oxo-′-10-apocarotenedialβ0-ionone ( ( ) ) 4,4′-diaponeurosporen-4′-oic acid NosCCD Apo-8 -10-apocarotenedial ( ) 4,4′-diaponeurosporen4-ketomyxol-2′-fucoside-4′-oic acid 13 Apo-8′-10-apocarotenedial ( )

3-OH-4-oxo-β-ionone ( ) 4-hydroxymyxol-2Myxol-2′0-fucoside′-fucoside NosCCDNSC3 Apo-10′-diaponeurosporenal ( ) 4-hydroxymyxol-2Myxol-2 -fucoside′-fucoside NSC3 ApoApo-83-OH-4-oxo--10′′--10-apocarotenedialdiaponeurosporenalβ-ionone ( ( )( )) NosCCD

4-hydroxymyxol-2′-fucoside Apo-8′-10-apocarotenedial3-OH-β -ionone( ) ( ) 3, 4-OH-β-ionone ( ) NosCCDNosCCD 3-OHApo-8-β-ionone0-10-apocarotenedial ( ) ( ) Apo-83,3′ -10-apocarotenedial4-OH--OH-ββ-ionone-ionone ( () ) ( ) 4-hydroxymyxol-2Myxol ′-fucoside NosCCD Apo-8′3-OH10-apocarotenedial-β-ionone ( ) ( ) Myxol NosCCD Apo-8Apo-3,8′ ′-10-apocarotenedial-4-OH-10-apocarotenedialβ-iononeacetate ( )adduct ( ) C10 39 NosCCD Apo-8′acetate-10-apocarotenedial adduct C10 ( ) 39 4-hydroxymyxol-2′-fucoside Apo-8′-10-apocarotenedial ( ) 30 3, 4-OH-β-ionone ( ) 0 NosCCD Symbols indicate4-ketomyxol-2 double bonds-fucoside where oxygenases cleave. : C9=C10; Apo-8 : C13=C14;3, ′4-OH--10-apocarotenedialβ-ionone : (C15=C15 ) ( ) ′; Symbols indicate double bonds where oxygenases cleave.NosCCD : C9=C10; : C13=C14; : C15=C15′; : C13′=C14′; :4 C11-ketomyxol′=C12-′2′; -fucoside: C9′=C10 ′; : C7′=C8′. Apo-8′-10-apocarotenedial ( ) Symbols: C1339′=C14 indicate ′; : C11 double′=C12 bonds′; : C9 where′=C10 oxygena′; : C7′ses=C8 cleave.′. : C9=C10; : C13=C14; : C15=C15′; 3-OH-4-oxo-β-ionone ( ) : C13 ′=C14′; : C11′=C12′; : C9′=C10′; : C7′=C8′. NosCCD 0 Symbols indicate doubleInt. J. bondsMol. Sci. where 2016 oxygena, 17, 1781ses cleave. : C9=C10; 3 : -OHC13=C14;Apo-8-4-oxo-β- ionone-10-apocarotenedial : C15=C15 ( ) ′; ( ) 9 of 37 NosCCD Symbols: C13′=C14 indicate′; : C11double′=C12 bonds′; : C9 where′=C10 oxygena′; : C7 ′ses=C8 cleave.′. : C9=C10; Apo : -C13=C14;8′-10-apocarotenedial : C15=C15 ( ) ′; : C13′=C14′; : C11′=C12′; : C9′=C10′; : C7′=C8′. Apo-12′- 0 cantaxanthinal ( ) 4-hydroxymyxol-2 -fucoside 1 4-hydroxymyxol-2′-fucosideAstaxanthin 3-OH-β-ionone ( ) Apo-13′- 3, 4-OH-3-OH, 4-oxo-β-iononeβ-inone ( )( ) astaxanthinone ( ) NosCCD 0 Apo-8 -10-apocarotenedialApo-10,10′-apocarotene ( ) Apo-14′- 3, 4-OH-β-ionone ( ) -dial ( ) astaxanthinal ( ) NosCCD References [19]Apo -8′-10-apocarotenedial ( [30,33]) [36] [31] [37] [38] [38]

Symbols indicate double bonds where oxygenases cleave. 0 : C7–C8; 0 : C9–C10;0 : C13–C14; : C15–C15′; : C13′–C14′; : C11′-C12′; : C9′–C10′; Symbols indicate double bonds where oxygenases cleave. : C9=C10; : C13=C14; : C15=C15 ; : C13 =C14 ; : C110=C120; : C90=C100; : C7 C7′0–C8=C8′0. .When an enzyme cleaves at different positions, the predominant product is indicated in bold. Lack of bold means no product preference or information Symbols indicate double bondsnot available. where oxygenases cleave. : C9=C10; : C13=C14; : C15=C15′; : C13′=C14′; : C11′=C12′; : C9′=C10′; : C7′=C8′.

3. Fungal Carotenoid Oxygenases Carotenoid production is a frequent trait in fungi. Best-known examples are the production of β-carotene in different taxonomic groups, neurosporaxanthin in ascomycetes, and astaxanthin in basidiomycetes. In some fungi, as Phycomyces blakesleeanus, Mucor circinelloides, Neurospora crassa, Fusarium fujikuroi, and Xanthophyllomyces dendrorhous, the genetic and biochemical basis of their carotenoid production has received considerable attention, and the functions and regulation of all the structural genes have been thoroughly investigated [2]. In addition, some fungi are excellent 1 carotenoid producers, and they have been adopted as biotechnological carotenoid sources [39,40]. In

contrast to photosynthetic organisms, in the cases investigated, the presence of carotenoids is dispensable, and the mutants unable to make carotenoids are viable. In some fungal models, such as N. crassa, such mutants have been widely used as easily traceable genetic markers. In fungi, carotenoid biosynthesis derives from the mevalonate pathway, as indicates the labeling of carotenoids with radioactive mevalonate [41,42]. The biosynthetic pathways are similar to those of photosynthetic species, except that a single desaturase is responsible for all desaturation steps from phytoene, and the cyclase and phytoene synthase activities depend on a single bifunctional gene. Several CCD enzymes have been identified in fungi participating in late steps of their carotenoid pathways. They are related to the production of three different compounds: retinal, neurosporaxanthin, and trisporic acids (Figure 2). 1

1 1

1

Int. J. Mol. Sci. 2016, 17, 1781 11 of 38 Int. J. Mol. Sci. 2016, 17, 1781 11 of 37

3. Fungal Fungal Carotenoid Carotenoid Oxygenases Oxygenases Carotenoid production production is is a a frequent frequent trait trait in in fu fungi.ngi. Best-known Best-known examples examples are are the the production production of βof-caroteneβ-carotene in indifferent different taxonomic taxonomic groups, groups, neurospo neurosporaxanthinraxanthin in in ascomycetes, ascomycetes, and and astaxanthin astaxanthin in basidiomycetes. In In some some fungi, fungi, as as Phycomyces blakesleeanus ,, MucorMucor circinelloides ,, Neurospora crassa ,, Fusarium fujikuroi, and Xanthophyllomyces dendrorhous ,, the the genetic genetic and and biochemical basis basis of their carotenoid production has received considerable attention,attention, and the functions and regulation of all the structural genesgenes have have been been thoroughly thoroughly investigated investigated [2]. In [2]. addition, In addition, some fungi some are fungi excellent are carotenoid excellent producers,carotenoid andproducers, they have and been they adoptedhave been as adopted biotechnological as biotechnologica carotenoidl sourcescarotenoid [39 ,sources40]. In contrast [39,40]. In to photosyntheticcontrast to photosynthetic organisms, in organisms, the cases investigated, in the cases the investigated, presence of carotenoids the presence is dispensable, of carotenoids and the is dispensable,mutants unable and to the make mutants carotenoids unable are to viable.make carotenoids In some fungal are viable. models, In such some as fungalN. crassa models,, such mutantssuch as N.have crassa been, such widely mutants used ashave easily been traceable widely used genetic as markers.easily traceable genetic markers. In fungi, carotenoid biosynthesis derives from the mevalonate mevalonate pathway, pathway, as indicates the labeling of carotenoids with radioactive mevalonate [[41,42].41,42]. The biosynthetic pathways are similar to those of photosynthetic species, except that a single desaturasedesaturase is responsible for all desaturation steps from phytoene, and the cyclase and phytoene synthase activities depend depend on on a a single bifunctional gene. gene. Several CCDCCD enzymesenzymes have have been been identified identified in fungi in participatingfungi participating in late stepsin late of theirsteps carotenoid of their pathways.carotenoid Theypathways. are related They to are the productionrelated to ofthe three prod differentuction of compounds: three different retinal, compounds: neurosporaxanthin, retinal, neurosporaxanthin,and trisporic acids (Figure and trisporic2). acids (Figure 2).

Figure 2. Enzymatic reactions achieved by fungal CCDs. (A) Retinal production from β-carotene by Figure 2. Enzymatic reactions achieved by fungal CCDs. (A) Retinal production from β-carotene by the the Cco1 (Ustilago maydis) and CarX (F. fujikuroi) CCDs; (B) β-apo-4′-carotenal production from Cco1 (Ustilago maydis) and CarX (F. fujikuroi) CCDs; (B) β-apo-40-carotenal production from torulene by torulene by the CarT (F. fujikuroi) and Cao-2 (N. crassa) CCDs; (C) β-apo-13-carotenone production the CarT (F. fujikuroi) and Cao-2 (N. crassa) CCDs; (C) β-apo-13-carotenone production from β-carotene from β-carotene by the sequential cleavage by the CarS and AcaA CCDs (P. blakesleeanus). Cleavage by the sequential cleavage by the CarS and AcaA CCDs (P. blakesleeanus). Cleavage sites are shaded sites are shaded and indicated by an arrow. and indicated by an arrow.

3.1. Fungal CCDs Involved in Retinal Production Rhodopsins are membrane photoreceptors using retinalretinal as a chromophore. The tertiary structure of these proteins is highly conserved and consistsconsists of seven transmembrane helices with a lysine residue to which an all-trans-retinal chromophore is covalentlycovalently bound. Rhodopsins are found in all major taxonomic groups, including fungi [43]. [43]. Very Very few fungal rhodopsins havehave been functionally investigated, amongamong them them NOP-1 NOP-1 in N.in crassaN. crassa[44], Ops[44], in OpsLeptosphaeria in Leptosphaeria maculans maculans[45,46], and [45,46], CarO [and47], CarOand OpsA [47], inandF. fujikuroiOpsA in[ 48F. ].fujikuroi The participation [48]. The participation of retinal in theof retinal activity in of the these activity proteins of these has only proteins been hasdemonstrated only been demonstrated for NOP-1 [49 ]for and NOP-1 CarO [49] [50]. and CarO [50]. Animals obtain retinal from β-carotene through its oxidative cleavage by a class of CCD enzymes [51]. A similar CCD, encoded by the gene carX [52], is used by F. fujikuroi to produce retinal from β-carotene (Figure 2) [53]. The gene carX is located in a coregulated cluster with the genes Int. J. Mol. Sci. 2016, 17, 1781 12 of 38

Animals obtain retinal from β-carotene through its oxidative cleavage by a class of CCD enzymes [51]. A similar CCD, encoded by the gene carX [52], is used by F. fujikuroi to produce retinal from β-carotene (Figure2)[ 53]. The gene carX is located in a coregulated cluster with the genes needed to produce β-carotene, carRA, and carB, and with the rhodopsin gene carO. Unexpectedly, despite the biochemical similarities between their carotenoid pathways, no carX ortholog has been identified in N. crassa. The only possible candidate for such activity in the genome of this fungus, the CCD CAO-1, was not active on carotenoid substrates, but cleaved efficiently the stilbene resveratrol [54]. A retinal-forming CCD enzyme, called Cco1, has also been described in the basidiomycete Ustilago maydis, producing minor amounts of β-carotene [55]. In this organism, retinal could be detected in cell extracts of the wild type and strains overexpressing cco1, but not in those of null cco1 mutants, and the in vivo β-carotene levels correlated inversely with the Cco1 activity. Retinal production seems to be particularly relevant in this fungus, as suggested by the occurrence of three genes in its genome for presumptive photoactive rhodopsins [55].

3.2. Fungal CCDs Involved in Trisporic Acid Production Trisporic acids, a class of fungal sexual hormones belonging to the trisporoids family, stand out among the compounds derived from β-carotene for their biological relevance [56,57]. Depending on small variations in their chemical structures, these hormones are distributed in five groups, named A, B, C, D, and E [56,58,59]. The synthesis, investigated in detail in B. trispora and other related species, requires the participation of two CCD enzymes. In fact, thorough chemical analyses revealed the generation of an unexpected apocarotenoid complexity in these fungi: B. trispora produces at least three groups of β-carotene derivatives—C18 trisporoids, C15 cyclofarnesoids, and C7 methylhexanoids [60]—and the same compounds were found in P. blakesleeanus cultures [61]. In the latter case, the origin from β-carotene was confirmed by their absence in a mutant unable to produce β-carotene. The first CCD enzymes known in fungi were Tsp3 and Tsp4 from Rhizopus oryzae, identified through the analysis of its genome and involved in trisporic acid biosynthesis [62]. This discovery led to identification of the tsp3 ortholog in B. trispora, whose connection with trisporic acids was reinforced by its induced expression by sexual interaction and its capacity to cleave β-carotene in a tsp3-expressing E. coli strain. Further evidence was obtained from the chemical structures of the apocarotenoids produced by P. blakesleeanus [63], which were consistent with the cleavage of β-carotene at the C110–C120 and C12–C13 double bonds (Figure2). The carS mutants of P. blakesleeanus, which accumulate large amounts of β-carotene, were found to be affected in a CCD enzyme [64], orthologous of Tsp3. The function of the gene, corroborated by the finding of relevant mutations in six different carS mutants, was confirmed by the capacity of the CarS protein to generate β-apo-120-carotenal (Figure2) in carS-expressing E. coli cells [65]. The identification of CarS as a CCD was unexpected, since the β-carotene over-accumulation phenotype of carS mutants suggested that the gene encoded a regulatory protein. This finding leads to the reinterpretation of the carotene overproduction of the carS mutation as a result of a blockage of the pathway or of the lack of a negative-acting apocarotenoid signal, which could be responsible for a formerly proposed feed-back regulation [66]. In support of the blockage hypothesis, the large increase in β-carotene content in these mutants is not sufficiently explained by the minor changes that could be found in the transcript levels of the structural genes carB and carRA [67]. However, the function of CarS as a CCD enzyme does not discard an additional regulatory role, as suggested by the unexpected albino phenotype of some double carS mutants [68]. Moreover, the carS mutants exhibit increased enzymatic activities in vitro [42], a result more coherent with a putative regulatory hypothesis. A second CCD enzyme, AcaA, has been characterized in P. blakeseleeanus. AcaA cleaves 0 β-apo-12 -carotenal (C25) to generate β-apo-13-carotenone (C18)[65]. The available information indicates that CarS and AcaA act sequentially to produce apocarotenoids in this fungus. First, CarS 0 0 cleaves β-carotene at the C11 –C12 double bond to generate the C25 and C15 apocarotenals, and AcaA Int. J. Mol. Sci. 2016, 17, 1781 13 of 38

cleaves the resulting C25-product at the C13–C14 double bond afterwards (Figure2). The resulting C 18 product is the origin of the trisporic acids and other trisporoids, while the former CarS C15 product is used for the synthesis of cyclofarnesoids. The genome of P. blakesleeanus contains three additional genes for presumptive CCDs. Only one of them contains the expected histidine residues to support CCD cleavage activity, but its function has not been investigated. The genes and enzymes for later steps of trisporoid metabolism are currently under study, but they are presumably CCD-unrelated enzymes: they include at least a 4-dihydromethyltrisporate dehydrogenase [69,70] and a 4-dihydrotrisporin-dehydrogenase [71], both identified in the P. blasleeanus relative Mucor mucedo.

3.3. Fungal CCDs Involved in Neurosporaxanthin Production

Neurosporaxanthin is a C35 carotenoid derived from the oxidative cleavage of its C40 precursor torulene. The first enzymatic reactions in the neurosporaxanthin biosynthetic pathway are similar to those for β-carotene production in other fungi, but in this case five desaturations instead of four and only one cyclization are introduced into the C40 carotene skeleton, leading to torulene (Figure2). The analysis of the proteomes of N. crassa and F. fujikuroi and the study of mutants blocked in different steps of the pathway led to the identification of the enzymes involved in the late oxidative reactions. The genomes of both species contain two CCD-encoding genes. One of them, called carT in F. fujikuroi, was found to catalyze the cleavage of torulene to produce β-apo-120-carotenal. This function was corroborated by the finding of a mutated carT allele in a reddish torulene -accumulating mutant and the ability of the wild-type carT allele to restore neurosporaxanthin production when it was introduced in the mutant [53,72]. CarT activity was also confirmed by targeted mutation in Gibberella zeae, a teleomorph of Fusarium graminearum [73]. The same function was achieved in N. crassa by its ortholog, cao-2, as demonstrated the accumulation of torulene in a mutant for this gene and the finding of cao-2 mutations in two torulene-producing mutants of this fungus [74]. As found for β-carotene production in Mucorales, the synthesis of neurosporaxanthin is induced by the light in N. crassa [75] and Fusarium sp. [76]. This photoresponse is achieved through an outstanding increase in mRNA levels for most of the structural genes, including cao-2 [74] in N. crassa, and carT [72] in F. fujikuroi. Moreover, the expression of carT is enhanced in carotenoid-overproducing mutants. The CarT and CAO-2 enzymes, catalyzing the asymmetrical cleavage of torulene at its acyclic end to remove a C5 segment, represent a novel CCD subgroup. The reaction is highly specific, as shown by the incapacity of CAO-2 to cleave the torulene precursor γ-carotene, indicating the need for five desaturations in the substrate molecule. Subsequently, the product β-apo-120-carotenal is converted to neurosporaxanthin by the aldehyde dehydrogenase YLO-1 in N. crassa [77] and by its ortholog CarD in F. fujikuroi [78].

4. Plant CCDs The oxidative cleavage of carotenoids in plants leads to the production of a range of apocarotenoids compounds that serve critical functions including photoprotection, photosynthesis, pigmentation, and signaling [79,80]. Plant CCDs constitute the most abundant group identified so far. These CCDs have been classified into two large families based on whether they are involved or not in the production of abscisic acid (ABA), a hormone involved in drought stress responses and in bad and seed dormancy [81]. Those involved in ABA production are the nine-cis-epoxy-carotenoid-dioxygenases (NCEDs), which cleave 9-cis-violaxanthin and 9-cis-neoxanthin to xanthoxin, the precursor of ABA. In fact, the first CCD cloned from any organism was the Vp14 gene from maize that catalyzed the first committed step in ABA biosynthesis [81]. After the identification of Vp14, other CCD enzymes were isolated, including β-carotene oxygenase (BCO) in mammals involved in the synthesis of vitamin A, CCDs from microorganisms (mentioned in former sections), and other plant CCDs. In plants, five CCD groups have been identified so far–CCD1, Int. J. Mol. Sci. 2016, 17, 1781 14 of 38

CCD2, CCD4, CCD7, and CCD8. However, transcriptome and large-scale genomic sequencing projects in many different plant species are allowing the identification of new species-specific CCDs with yet unknown functions. In fact, biochemical studies have been reported for a limited number of plant CCDs, and the roles they play in different organisms are not fully understood.

4.1. CCD1 and CCD2 Subfamilies AtCCD1 from A. thaliana was the first carotenoid cleavage dioxygenase isolated not involved in ABA biosynthesis (Figure1)[ 82]. The first cleavage activity reported for CCD1 enzymes was on the 0 0 C9–C10 (C9 –C10 ) double bonds in C40 carotenoids, producing a colored C14 dialdehyde and two scent C13 products [82]. Homologues of this enzyme have been identified in many other plant species [83–91], and the studies performed on these CCD1 enzymes suggested additional cleavage activities on acyclic, monocyclic, and bicyclic carotenoids, such as ζ-carotene, lycopene, phytofluene, β-carotene, δ-carotene, zeaxanthin, lutein, and violaxanthin, and on different apocarotenoids, catalyzing the symmetric cleavage at C9–C10 (C90–C100), C5–C6 (C50–C60), C7–C8 (C70–C80), and C13–C14 (C130–C140) (Figure3)[ 85,92–95]. The enzyme cleaved symmetrically at C9–C10 (C90–C100) of acyclic and cyclic trans-carotenoids and did not cleave adjacently to a -cis double bond or an allenic bond found in some carotenoids [82]. Therefore, an asymmetric cleavage is observed in such cases. The C5–C6 or 0 0 C5 –C6 activity of CCD1 enzymes on lycopene (or both), leading to the formation of the C8 ketone 6-methyl-5-hepten-2-one (MHO), was reported for the first time in tomato, maize, and A. thaliana [92] and was later detected in Cucumis melo [84], Rosa damascena [96], Oryza sativa [97], and Vitis vinifera [98]. The cleavage of C7–C8 and C70–C80 double bonds of linear and monocyclic carotenoids constitutes a novel recognition site for the CCD1 plant subfamily. So far only detected by in vitro assays for the rice CCD1 enzyme [85], this activity allowed for the formation of C10-aldehyde geranial, suggesting an alternative pathway for the geranial formation in plants.

4.1.1. Mode of Action and Functional Implications Initially, the CCD1 enzymes were suggested to be involved in the biosynthesis of apocarotenoid volatiles, such as geranylacetone, pseudoionone, and β-ionone [79], which possess an extremely low threshold for human perception [99]. Lowering of CCD1 expression in tomato or petunia led to significant reductions in the emission rates of β-ionone and geranylacetone, but did not lead to significant changes in the carotenoid concentration [90], suggesting additional roles for the CCD1 enzymes. In addition, the expression of CCD1 during tomato, strawberry, melon, and grape development, or in saffron flowers, did not mirror the emission of apocarotenoid volatiles [83,84,86,89,90]. Furthermore, experiments conducted to identify loci affecting volatile composition in tomato did not reveal the implication of CCD1 in the emissions of apocarotenoid volatiles [100]. These are produced from C40 carotenoids localized in plastids, and the CCD1 enzymes lack plastid targeting signals and are cytoplasmatic [87,101], suggesting that CCD1 enzymes mainly act in planta as scavengers of carotenoid degradation products of different chain lengths rather than primary cleavers of intact carotenoids [80,85,93,94,98,102]. The key roles of carotenoids in photosynthesis, photomorphogenesis, and plant development suggest that their biosynthesis and degradation is coordinately regulated with processes such as plastid biogenesis, flowering, and fruit development [103]. In addition, the link of carotenoid biosynthesis with those of gibberellin, ABA, and strigolactone phytohormones implies that changes in the composition or content of carotenoids might bring about physiological or biochemical shiftings in plants [4]. In 2008, a role of CCD1 in the production of strigolactones (SLs) during the arbuscular mycorrhiza symbiosis in roots was described [94]. CCD1 cleaves the C27 apocarotenoid derivatives produced by the activity of CCD7, which is also involved in the biosynthesis of SLs [104], producing C13 α-ionol and C14 mycorradicin, which are indicators of a well-established and functional symbiosis [105], reducing SL production and avoiding over-colonization [106]. Int. J. Mol. Sci. 2016, 17, 1781 15 of 38

CCD1 could be also important in plant stress responses. Enhanced CCD1 expression during berry development has been associated with the increased osmotic stress that occurs during ripening, resulting in leaky membranes and concomitant chloroplast degradation [98]. In addition, CCD1 expression is induced during leaf senescence [89,107], suggesting that the enzyme-mediated degradation of carotenoids, apocarotenoids, or both is catalyzed by cytosolic CCD1 enzymes. Furthermore, plant–insect interactions, extreme temperatures, high irradiance, or ultraviolet (UV) stress induceInt. theJ. Mol. production Sci. 2016, 17, 1781 of β-ionone and β-cyclocitral among other apocarotenoid volatiles15 [of108 37 ,109]. Int. J. Mol. Sci. 2016, 17, 1781 15 of 37 CCD1 expression has been detected in all tested tissues in different plant species [89–91,95,98,110,111], CCD1 expression has been detected in all tested tissues in different plant species [89–91,95,98,110,111], but its expression is stimulated by abiotic stress or ABA treatment [112,113]. butCCD1 its expression expression is stimulatedhas been detected by abio intic all stress tested or tissuesABA treatment in different [112,113]. plant species [89–91,95,98,110,111], but its expression is stimulated by abiotic stress or ABA treatment [112,113].

Int. J. Mol. Sci. 2016, 17, 1781 8 of 37

Table 2. Enzimatic activity and substrate specificity of different bacterial carotenoid oxygenases.

Enzymes and Products Assayed Substrates β-carotene-oxygenase NosCCD (NSC1) MtCCO Mycobacterium NSC3 Nostoc sp. NACOX1 SaCCO Sphingopyxis PpCCO Plesiocystis Microcystis PCC 7806 Nostoc sp. PCC 7120 tuberculosis PCC 7120 Novosphingobium alaskensis pacifica β-ionone ( ) + Apo-8,10′ β-apo-13-carotenone Apo-12′- β-apo-8′-carotenal -apocarotene-dial ( ) β-apo-13-carotenone () () β-apo-13- carotenal ( )

Other minority products Retinal ( ) β-apo-14′-carotenal carotenone ( ) Apo-10′- ( , ) Transretinal ( ) carotenal ( ) β-apo-10′-carotenal β-ionone ( ) + Apo-10,10′ β-apo-13-carotenone ()

-apocarotene-dial ( ) Retinal ( )

3-OH-β-apo-8′-carotenal 3-OH-β-ionone ( ) + Apo- 3-OH-β-apo-13-carotenone ( ) 8,10′-apocarotene 3-OH-retinal () -dial ( ) 3-OH-β-apo-10′-carotenal 3-OH-β-ionone ( ) + Apo- 3-OH-β-apo-13-carotenone ( ) 10,10′-apocarotene 3-OH-retinal () -dial ( ) γ-carotene β-ionone ( ) + Apo-8,10′ β-apo-13-

-apocarotene-dial ( ) carotenone ( )

β-carotene β-apo-13-carotenone ( ) 2 × β-cyclocitral ( , ) Apo-10,10′-apocarotene Retinal ( ) No No No (in vitro) No Crocetindial ( , ) -dial ( ) β-apo-14′-carotenal ( ) Apo-13′- 2 × hydroxi-β- 3-OH-β-apo-13-carotenone ( ) zeaxanthinone ( ) Zeaxanthin 3-OH-β-ionone ( ) cyclocitral 3-OH-β-apo-14′-carotenal ( ) Apo-14′- Apo-10,10′-apocarotene Int. J. Mol.No Sci. 2016, 17, 1781 No No (in vitro) 9 of 37 (, ) 3-OH-retinal ( ) zeaxanthinal ( ) -dial ( ) Int.Int. J.J. Mol.Mol. Sci.Sci. 20162016Int.,, 1717 J., ,Mol. 17811781 Sci. 2016, 17, 1781 Crocetindial ( , ) 3-OH-β-apo-11-carotenal ? 99 ofof 37Apo-1237 ′- 9 of 37 Apo-12′- zeaxanthinal ( ) cantaxanthinal ( ) Lycopene Apo-12Apo-12′-′- Yes (in Apo-12vivo) ′- No Astaxanthin No poorly 3-OH-β-ionone ( ) Apo-13′- cantaxanthinalcantaxanthinal Unknown( ( ) ) cantaxanthinal product ( ) 3-OH, 4-oxo- β-inone ( ) astaxanthinone ( ) AstaxanthinAstaxanthin Astaxanthin 3-OH-3-OH-ββ-ionone-ionone ( ( ) ) 3-OH-β-ionone ( ) Apo-13Apo-13′-′- Apo-13′- Figure3-OH-β -apo-13-carotenone3. Activity of plant( ) CCD1 and CCD2 subfamilies, showing their divergenceApo-10,10 in′ -apocarotenesubstrate Apo-14′- 3-OH,3-OH, 4-oxo- 4-oxo-ββ-inone-inone3-OH, ( ( ) ) 4-oxo-βFigure-inone (3-OH- )3. FigureActivityβ-apo-14 3. ′-carotenalActivity of plant ( of ) plant CCD1 CCD1 and and CCD2 CCD2 subfamilies, subfamilies, showing showingastaxanthinoneastaxanthinonetheir their divergencedivergence ( ( ) ) astaxanthinone in in substrate substrate ( ) Lutein ζ -dial ( ) astaxanthinal ( ) Apo-10,10Apo-10,10′-apocarotene′-apocaroteneApo-10,10 ′-apocarotenespecificity and cleavage sites on the following substrates: (A) -carotene andApo-14Apo-14 lycopene;′-′- (B) Apo-14β-carotene;′- specificityspecificity and3-OH-retinal cleavage and ( cleavage ) sites on sites the on following the followingReferences substrates: substrates:No (A ()Aζ)-carotene ζ-carotene[19] and and lycopene;lycopene; [30,33] ( (BB) )β β-carotene;-carotene; [36] [31] [37] [38] [38] -dial-dial ( ( ) ) -dial ( ) and (C) zeaxantine. Symbols indicate double bonds where oxygenasesastaxanthinalastaxanthinal cleave. ( :( C5–C6; ) ) astaxanthinal : C7–C8; ( ) 3-OH-α-apo-15′-carotenal References [19] [30,33] [36] and (C) zeaxantine.[31] Symbols Symbolsindicate[37] double indicate bonds double where[38] bonds oxygenases where oxygenases cleave. [38] : C5–C6;cleave. : :C7–C8; C7–C8; : C9–C10; : C13–C14; : C15–C15′; : C13′–C14′; : C11′-C12′; : C9′–C10′; References References [19] [19] [30,33] [30,33]and ([3C6]:) C9–C10 zeaxantine. and() C9[3 Symbols6]′ –C10[31]′ ; indicate : C13–C14;[31] double [37] : C13 bonds′–C14′; where[37]:[38] C11 ′ oxygenases–C12′. [38] cleave. [38] : C5–C6; [38] : C7–C8; 0 0 0 0 0 0 Symbols indicate double bonds where oxygenases cleave. 4-oxo- : C7–C8;β-ionone ( :) C9–C10;: C9–C10 : C13–C14; and C9 ′–C10 : C15–C15′; : C13–C14;:′ ;C7 ′–C8: C13 ′. When :′ –C14C13 ′–C14an′; enzyme:′ ; C11: C11′-C12 cleaves′–C12′; at′.: differentC9′–C10′ ;positions, the predominant product is indicated in bold. Lack of bold means no product preference or information Symbols indicateSymbols Echinenonedouble indicate bonds wheredouble oxygenases bonds where cleave. oxygenases : C7–C8; cleave. : :C9–C10; C9–C10 : C7–C8; and : C13–C14; :C9 C9–C10;–C10 ; :: C15–C15 C13–C14;C13–C14;′; : : C13C13C15–C15′–C14–C14′; ; :: C11 C11C13′′-C12–C14–C12′;′ ; . :: C9C11′–C10′-C12′; ′; : C9′–C10′; Apo-10,10′-apocarotene :: C7C7′′–C8–C8′′.. WhenWhen : an anC7 enzyme′enzyme–C8′. When cleavescleaves an enzyme atat differentdifferent cleavesNo positions,positions, at different thethe predominantpredominant positions, the 4.1.2. productproduct predominant CCD1 isis indicatedindicated Gene product Family inin bold.bold. is indicated and LackLack Genomic ofof in boldboldnot bold. available. meansmeans Organization Lack nono of productproductbold means prpreferenceeference no product oror information information preference or information -dial ( ) 4.1.2. CCD1 Gene Family and Genomic Organization notnot available.available. not available. β-ionone4.1.2. ( ) CCD1 Gene Family and Genomic Organization Canthaxantin The CCD1 family is present in some plant species as a multigene family (Table S1), and studies The CCD1 family is present in some plant species as a multigene familyApo-13 (Table′-canta- S1), and studies 2 x 4-oxo-β-iononeon ( ) maize and tomato CCD1 enzymes have suggested varying expression profiles and different The CCD1 family is present in some plant species as a multigene family (Tablexanthinone S1), ( ) and studies on Apo-10,10′-apocaroteneon maize and tomato CCD1 enzymes have suggest ed varying expression profiles and different specificities in their activity towards different substrates and in their double Apo-14bond′- preferences -dialmaize ( ) and tomato CCD1 enzymes have suggested varying expression profiles and different specificities [90,92,93,102,114].specificities in Intheir most activity of the analyzedtowards species,different there substrates are two and genes in encodingtheir doublecantaxanthinal for CCD1 bond ( )enzymes, preferences in their activity[90,92,93,102,114]. towards In different most of substratesthe analyzed and species, in their there double are two bond genes preferences encoding for [ 90CCD1,92,93 enzymes,,102,114 ]. In mostand ofin many the analyzed cases the genes species, are present there arein tandem two genes in the encodingsame chromosome for CCD1 (Table enzymes, S1). The identities and in many amongand thein many paralogous cases the CCD1 genes genes are present range between in tandem 90% in theand same 99%. chromosome All the CCD1 (Table genes S1). show The several identities intronsamong in their the paralogous sequence [115], CCD1 these genes positions range betweenbeing conserved 90% and [115]. 99%. Moreover, All the CCD1 the presencegenes show of such several intronsintrons allows in their for thesequence generation [115], of these different positions splice be variantsing conserved (Table [115]. S1). These Moreover, variants the maypresence differ of as such wellintrons in the allowsuntranslated for the regions, generation with of implications different splice in transcript variants (Tablestability, S1). localization, These variants translation, may differ or as a combinationwell in the untranslatedthereof. It is likely regions, that with the truncatedimplications proteins in transcript from the stability, CCD1 mRNA localization, isoforms translation, may act or a combination thereof. It is likely that the truncated proteins from the CCD1 mRNA isoforms may act Int. J. Mol. Sci. 2016, 17, 1781 16 of 38 cases the genes are present in tandem in the same chromosome (Table S1). The identities among the paralogous CCD1 genes range between 90% and 99%. All the CCD1 genes show several introns in their sequence [115], these positions being conserved [115]. Moreover, the presence of such introns allows for the generation of different splice variants (Table S1). These variants may differ as well in the untranslated regions, with implications in transcript stability, localization, translation, or a combination thereof. It is likely that the truncated proteins from the CCD1 mRNA isoforms may act as dominant-negative regulators. In fact, several reports provide evidences of a dominant regulatory role for truncated proteins generated by splice variants in plants [116]. AtCCD1 has been suggested to act as a dimer [82], and the presence of these variants could be part of a regulatory mechanism for CCD1 activity. The phylogenetic analysis of the CCD1 proteins reveals several main clusters that fit with the phylogenetic relationships in embryophytes (Figure S1). In the angiosperm group, there are three sub-clusters corresponding to monocots, dicots (excluding crucifers), and crucifers. The latter sub-cluster of Brassicaceae species is more related to the monocot group than to the dicot group, suggesting a specific function for CCD1 in these plants.

4.1.3. Mode of Action of CCD2 Recently, a close CCD subfamily related to the CCD1 family has been identified in Crocus species [117,118]. This subfamily, named as CCD2 (Figure1), is involved in the formation of the apocarotenoid crocetin, which accumulates in stigma tissue at high levels [119]. Crocetin is derived by bio-oxidative cleavage of zeaxanthin by a C7–C8 and C70–C80 cleavage reaction catalyzed by CsCCD2 [117]. The expression of CsCCD2 is restricted to the stigma in saffron, and it mirrors the levels of crocetin in this tissue during its development [89,117]. The same behavior is observed for CaCCD2, a CCD2 homologue isolated from the spring crocus C. ancyrensis [118]. Both CCD2 enzymes are plastidic, a major difference with the CCD1 subfamily [118]. No other CCD2 homologues have been identified in other organisms, presumably due to the uncommon ability to synthesize crocetin in plants [120] or bacteria [121]. Besides zeaxanthin, CCD2 is able to recognize and cleave lutein and 3-OH-β-apocarotenals at the C7–C8 position, but it does not cleave β-carotene, lycopene, or β-cryptoxanthin [117].

4.1.4. Structure of CCD1 and CCD2 Enzymes Based on the crystal structure of VP14 [122], structural models have been built for CCD1 and CCD2. VP14 is a globular protein that showed a characteristic hydrophobic patch formed by the antiparallel α-helices presumably involved in membrane interaction [28]. CCD1 and CCD2 enzymes display from 58% to 39% overall identity with VP14. Specially conserved are the structural elements of the CCD enzymes. Three regions have been identified in VP14, implicated in the recognition of the bond to be cleaved [122]. The first one is Val478, which is a substitute for a Phe residue in CCD1 and CCD2 enzymes. This substitution is present in many others CCDs. The second region is a loop formed by residues 499 to 503 in VP14. This loop is not present in CCD1 and CCD2 enzymes, in which conserved Leu170 in Vp14 was replaced by Trp. In Vp14, the loop and the leucine residue form a pocket where the second ring of the carotenoid accommodates in such a way that the C11–C12 double bond of the substrate should be close to the Fe2+. The third region, located in the active center, comprises three Phe residues in Vp14 (Phe171, Phe411, and Phe589) and is conserved in all NCED and CCD1 enzymes; however, in CCD2, only one Phe residue is conserved.

4.1.5. Regulation of CCD1 and CCD2 Enzymes As stated before, CsCCD2 expression is regulated during the development of the stigma and the enzyme is localized in chromoplasts [123] where catalyzes crocetin biosynthesis. Crocetin is oxidated and glucosylated thereafter [124], generating crocins that accumulate in the vacuole. The mechanisms involved in CsCCD2 expression and activity have recently been elucidated [123]. Int. J. Mol. Sci. 2016, 17, 1781 17 of 38

In this study, the authors showed that high temperatures repressed CsCCD2 expression, while low temperatures have an opposite effect by upregulating its expression. In saffron plants, the highest expression levels of CsCCD2 are related to the lowest temperatures. Several classes of cis-regulatory elements were identified in the promoter of CsCCD2. The regulation of carotenoid biosynthesis by light and dark conditions has been investigated in red pepper [125], in tomato leaves [126], and in citrus, where the expression levels of the carotenogenic genes are affected by circadian rhythms [127,128]. Additionally, a new mechanism of regulation by alternative splicing has been observed for CsCCD2,[123]. The obtained data agree with the predicted function of alternative splicing in genes with a circadian clock expression pattern, where oscillations of protein expression levels require rapid adjustments in mRNA levels over the course of a day [129].

4.2. The CCD4 Subfamily The CCD4 subfamily is present in all flowering plants, usually composed by several members (Figure1)[ 115,130]. Their functions have been directly linked to coloration and production of aroma volatiles in flower and fruit tissues [89,98,131–133]. More recently, CCD4 activity has been reported in seeds, leaves, and roots associated with carotenoid turnover [107,134] or with the production of novel apocarotenoid-derived signals [135]. The first member of this subfamily was identified in A. thaliana [18]; however, its role in carotenoid catabolism was initially described for CCD4a from Chrysantemum morifolium, whose activity is responsible for the absence of carotenoid accumulation in petals of white flowers [136]. All CCD4 proteins contain a plastid target peptide; moreover, suborganellar studies in different plant species localized these enzymes in the plastoglobuli together with other carotenoid biosynthetic enzymes, which allows direct access to their substrates [131,137–141]. In both chloro- and chromoplasts, CCD4 is one of the major components of plastoglobuli proteomes [137,140], indicating a role for this enzyme in the regulation of carotenoid metabolism in plastid types with marked differences in functionality and carotenoid composition [142]. Moreover, CCD4 protein level is strongly reduced in Arabidopsis mutants defective in the ABC1K1 and ABC1K3 plastoglobuli kinases, suggesting phosphorylation as a post-transcriptional modification necessary for CCD4 protein stability and supporting main activity and localization in plastoglobuli [143].

4.2.1. Enzyme Structure and Functional Implications As found for other CCDs, molecular modeling of CCD4 enzymes from the structure of the 9-cis-epoxycarotenoid dioxygenase from maize (VP14/NCED) revealed a well-conserved structure among the CCD family [130,133]. The analyzed CCD4s display two main functional domains: the helical domain composed of two antiparallel α-helices, which might penetrate in the hydrophobic core of the plastoglobule where carotenoids accumulate [130,138], and the β-propeller structure that forms a long central tunnel, where Fe2+ is coordinated with four conserved His in the active center [130,133]. However, other critical amino acids for NCED activity are not always conserved among all CCD4 members [130,133]. As was the case in CCD1 and CCD2 enzymes, a large number of CCD4 proteins display a Phe residue instead of the conserved Val478 found in NCED, with the exception of CCD4 from alfalfa (CCD4a and b), grape (CCD4e), potato (CCD4b), poplar (CCD4c), and citrus (CCD4b1), where it is substituted by Leu; however, in citrus CCD4c, Val478 is replaced by Met. Other critical residues in VP14 are three Phe at positions 171, 411, and 589, from which only Phe589 is conserved in all CCD4, while Phe411 is highly variable, and Phe171, which also takes part of the motif FDG, is partially conserved and usually substituted by Leu. Another important region for VP14 activity is a loop located on the back side of the substrate pocket, involving residues 499–503 and Leu170. In contrast with CCD1 and CCD2, residues in this loop are highly conserved in the CCD4 subfamily; however, the citrus CCD4b1 is a relevant exception, with the Glu499 and Pro500 reported in VP14 substituted by Lys and Glu, respectively. The Leu170 in VP14 is also well conserved in all CCD4s; however, in CCD4b1, CCD4b, and CCD4e from citrus, potato, and poplar, it is replaced by Int. J. Mol. Sci. 2016, 17, 1781 18 of 38

Phe and in citrus CCD4c by Pro. Although it is difficult to determine exactly the impact of these residue changes in the enzyme activity, it is likely that they may modulate the carotenoid substrate specificity, the cleavage position, or both. Interestingly, functional assays performed with citrus CCD4b1, which accumulates several non-conservative amino acid substitutions, show an unusual cleavage position, having as preferential substrates the hydroxylated β-ionone ring of the xanthophylls. Future studies on comparative 3D modeling of the different members of this subfamily will provide clues as to substrate specificity or cleavage position in carotenoid backbone, which may help to understand their role in vivo.

4.2.2. Gene Family and Genomic Organization The number of genes for CCD4 enzymes is very variable among different species, with at least two genes identified in most plants [115,144]. The presence of one single CCD4 gene has been reported only in a limited number of plant species with an available genome sequence, such as A. thaliana, Carica papaya (papaya), Prunus persica (peach), and Sorghum bicolor (sorghum) [115,130,144]. By contrast, the largest number of CCD4 genes has been found in Citrus sp. (a, b1, b2, c and d), Populus trichocarpa (a–e), and Chrysanthemum morifolium (a-1–a-4 and b), with five members and with at least one of them being a putative pseudogene [115,133,145,146]. Four members have been identified in Vitis vinifera (grapevine) (a, b, c, and e) or Brassica napus (rapeseed) (A1, A8, C1, and C3), while three are present in Theobroma cacao (cacao) [98,130]. Another common genomic feature of CCD4 genes is the low presence of introns, most of them being intron-less or containing a single intron [115,144]. Moreover, intron size and position are quite variable among different species. A comprehensive comparative analysis of the genomic structure of CCD4 from diverse plants showed that introns are located in three different sites and only those located at the 30 end of the gene maintain a conserved position [115]. Exceptionally, only a few CCD4 genes contain more than one intron, such as Osmathus fragans CCD4 and Solanum lycopersicum CCD4a and b, each with two introns, Citrus sp. CCD4d with two or four introns, and an uncharacterized CCD4 from apple (Malus domestica) with 85% identity at the protein level to apple CCD4a [96] with six introns. The degree of protein sequence identity among CCD4 members is quite variable, ranging from 30% to 98%, with typical values around 50%–70% [115,130,133,147,148]. In multigene CCD4 families, a high degree of homology (85%–98% identity at the protein level) is usually found between the different members, frequently located in tandem on the same chromosome (i.e., tomato CCD4a and b, citrus CCD4b1 and b2, chrysamthemum CCD4a-1 to a-4, or Populus CCD4c to e)[115,133]. As expected, the highest variability is found at the plastid target peptide sequences, while a higher conservation is found in the motifs described as essential for CCD activity [6,28,149]. The moderate conservation within the different CCD4 members of the same family (i.e., citrus CCD4b1 versus CCD4c or grapevine CCD4a/b versus CCD4c/e), and the location in different clusters in the phylogenetic analysis (Figure S2) suggest a divergent evolution and functionality of these members.

4.2.3. CCD4 Activity: Carotenoid Substrates and Cleavage Products In contrast to other CCD subfamilies, such as NCED or CCD7/8, functional assays of CCD4 indicate that the members are rather heterogeneous in respect of carotenoid substrates and cleavage positions (Table4). The functional characterization of CCD4 has been an active field of research in the last decade by following three basic strategies: (i) in vivo assays of CCD4-defective plant mutants or CCD4-overexpressing or repressing transgenic plants; (ii) in vivo assays of bacteria over-accumulating different carotenoids and co-expressing CCD4; and (iii) in vitro enzymatic assays performed with recombinant CCD4. Table4 summarizes the information currently available on CCD4 enzyme activity. The first comparative study of substrate preferences and cleavage positions of CCD4 enzymes was obtained from in vitro and in vivo assays performed with five CCD4s isolated from Arabidopsis, rose (R. damascene), osmanthus (O. fragans), apple (M. domestica), and chrysanthemum (C. morifolium)[96]. Most of these CCD4 cleaved β-carotene at C9–C10 and C90–C100 positions, Int. J. Mol. Sci. 2016, 17, 1781 19 of 38

rendering C13 β-ionone, while no activity was detected on hydroxylated xanthophylls [96]. Further research using Arabidopsis ccd4 mutants or carotenoid over-accumulating lines revealed that epoxy-xanthophylls (mainly violaxanthin) are likely the substrates in Arabidopsis leaves [107,134] 0 0 to render C13 apocarotenoids (cleavage at C9–C10 or C9 –C10 double bonds) that undergo further glycosylation [134]. Interestingly, changes in carotenoid composition and apocarotenoid profile in ccd4 Arabidopsis mutants, during seed maturation or in roots of hyperaccumulating β-carotene plants, suggest that CCD4 also accepts β-carotene as substrate [107,134]. One of the two CCD4s from potato, in this work designated as CCD4a (Figure S2), was functionally characterized. Analysis of the carotenoid profile in tubers from RNAi CCD4 potato lines suggested violaxanthin as the main substrate [150]. However, in-depth biochemical characterization of potato CCD4a using in vitro and in vivo assays points to β-carotene as its main substrate, cleaved at C9–C10 or C90–C100 positions [148]. In saffron, in vivo assays showed that CCD4a and b cleave β-carotene and zeaxanthin most likely at the C9–C10 and C90–C100 double bonds, while CCD4c cleaves other xanthophylls such as lutein [89,130,151]. In vivo assays of grapevine CCD4a and b indicate that these enzymes, in contrast to all CCD4s characterized so far, do not cleave cyclic carotenoids, with the exception of β-carotene, cleaving linear carotenes neurosporene, lycopene, and β-carotene—only CCD4b preferentially at C9–C10 and C90–100 positions [98]. The carotenoid profile from rape (Brassica napus) flowers suggests that the member CCD4_C3 cleaves α-carotene or δ-carotene at the C9–C10 position to render α-ionone [152]. Interestingly, in vitro and in vivo assays show that Citrus sp. CCD4b1/CCD4 accepts β-ring hydroxylated xanthophylls as preferential substrates, but also accepts β-carotene or α-carotene. In contrast to the other plant CCD4s, citrus CCD4b1 and CCD4 cleaves the carotenoid backbone at C7–C8 or C70–C80 double bonds, resembling CCD2 activity, but only at one side of the molecule [131,133]. In summary, in vitro and in vivo assays for most of the CCD4 enzymes point to β-carotene and a C9–C10 or C90–C100 double bond cleavage as a favorite substrate and positions, respectively, although some relevant exceptions, such as citrus or grape CCD4, have been identified regarding the substrate or cleavage site. In particular, the exclusive cleavage activity reported for citrus CCD4b1 and CCD4, together with its restricted expression pattern, may indicate that this enzyme belongs to a novel class of CCD more functionally related to the recently characterized CCD2 from saffron [117,118]. In planta data, derived from the analysis of apocarotenoid profiles, the alteration in carotenoid complement in ccd4 mutants and overexpressed or repressed CCD4 plants, or both, point to xanthophylls as the preferential substrates in vivo [130,133,134,145]. Nevertheless, in some species and tissues, such as potato tubers, chrysanthemum petals, and peach fruit, β-carotene cannot be ruled out as the primary target since alterations in the xanthophylls composition in defective or overexpressing CCD4 plants can be explained by a reduction or increase in β-carotene content as the precursor of β-xanthophylls [136,150,153]. These differences in CCD4 substrate preferences may also reflect the differences in substrate availability or accessibility depending on the types of assay. In the in vitro and in vivo assays, the enzyme has direct access to the potential substrates; this clearly differs from the in planta situation, where carotenoids are integrated in the suborganellar structures of the plastids, and CCD4 is preferentially associated with plastoglobuli, where xanthophylls are synthesized and accumulated. The CCD4 activity on linear carotenes is usually very weak either in vivo or in vitro, with the exception of grapevine CCD4 [98]. Recently, the phenotypic characteristics of the Arabidopsis double mutant clb5 ccd4 (defective in ZDS and CCD4) suggests that, under this particular scenario of hyper-accumulation of linear upstream carotenes, CCD4 generates an apocarotenoid derived from phytofluene, ζ-carotene, or both, indicating an activity on the backbone of these carotenes [135]. None of the CCD4 characterized so far catalyzes the cleavage of phytoene, in agreement with the hypothesis that a double bond in the carotenoid structure must be adjacent to the cleaved double bond [92,98]. Int. J. Mol. Sci. 2016, 17, 1781 20 of 38

Table 4. Functional characterization of CCD4 enzymes.

Species Protein Assay a Parent Carotenoid Cleavage Position Product Detected References in planta β-carotene, Violaxanthin n.d. n.d. [107] in planta Phytofluene, ζ-carotene n.d. n.d. [135] 0 0 Arabidopsis thaliana CCD4 in planta (leaf) Epoxy-β-xanthophylls C9–C10 or C9 –C10 C13-glycosids [134]

in planta (root) β-carotene n.d. long-chain free apocarotenals (C15 to C30)[134] in vitro Apo-β-caroten-80-al C9–C10 β-ionone [96] Brassica napus CCD4_C3 in planta α-carotene, δ-carotene C9–C10 α-ionone [152] Chrysantemum morifolium CCD4a in vivo and in vitro β-carotene C9–C10 (C90–C100) β-ionone [96] β-carotene, β-cryptoxanthin, 3-OH-apo-β-8-carotenal, apo-β-8-carotenal (C ); Citrus clementina CCD4b1 in vitro C7–C8 or C70–C80 30 [133] Zeaxanthin, Lutein, α-carotene β-cyclocitral and 3-OH-β-cyclocitral (C10) Citrus unshiu CCD4 in vitro and in vivo β-cryptoxanthin, Zeaxanthin C7–C8 or C70–C80 3-OH-apo-β-8-carotenal [131] CCD4a/b in vivo β-carotene, Zeaxanthin C9–C10 (C90–C100) β-ionone, β-OH-ionone [89,147] in vivo C9–C10 or C90–C100 β-carotene β-ionone, β-cyclocitral [130] Crocus sativus in planta C7–C8 or C70–C80 CCD4c Megastigma-4,6,8-triene in planta Lutein C9–C10 [130] (derived from 3-OH-α-ionone) Malus domestica CCD4 in vivo β-carotene C9–C10 (C90–C100) β-ionone [96] Osmanthus fragans CCD4 in vivo β-carotene C9–C10 (C90–C100) β-ionone [96] in vivo β-carotene C9–C10 (C90–C100) β-ionone [96] Rosa damascena CCD4 in vitro apo-β-8-carotenal C9–C10 β-ionone [96] in planta Violaxanthin n.d. n.d. [150] in vitro and in vivo β-carotene C9–C10 or C90–C100 Apo-β-caroten-100-al; β-ionone [153] Solanum tuberosum CCD4 α-carotene, Lutein, Zeaxanthin, 3-OH-β-apo-100-carotenal, in vitro C9–C10 or C90–C100 [153] β-cryptoxanthin β-apo-100-carotenal3-OH-ε-apo-10´-carotenal CCD4a/b in vivo ε-carotene, C9–C10 (C90–C100) α-ionone, [98] CCD4a/b in vivo Neurosporene C90–C100 Geranylacetone [98] Vitis vinifera CCD4a/b in vivo Lycopene C5–C6 (C50–C60) 6-methyl-5-hepten-2-one [98] CCD4b in vivo ζ-carotene C9–C10 (C90–C100) α-ionone, Geranylacetone [98] a In planta refers to data inferred from changes in carotenoid and/or apocarotenoid profiles in overexpressing or knockout CCD4 mutants; in vivo assay indicates data obtained from bacteria over-accumulating carotenoids and expressing CCD4; in vitro assay refers to enzymatic assays performed with recombinant CCD4 enzyme. n.d. not determined. Int. J. Mol. Sci. 2016, 17, 1781 21 of 38

These differences in CCD4 substrate preferences may also reflect the differences in substrate availability or accessibility depending on the types of assay. In the in vitro and in vivo assays, the enzyme has direct access to the potential substrates; this clearly differs from the in planta situation, where carotenoids are integrated in the suborganellar structures of the plastids, and CCD4 is preferentially associated with plastoglobuli, where xanthophylls are synthesized and accumulated. The CCD4 activity on linear carotenes is usually very weak either in vivo or in vitro, with the exception of grapevine CCD4 [98]. Recently, the phenotypic characteristics of the Arabidopsis double mutant clb5 ccd4 (defective in ZDS and CCD4) suggests that, under this particular scenario of hyper-accumulation of linear upstream carotenes, CCD4 generates an apocarotenoid derived from phytofluene, ζ-carotene, or both, indicating an activity on the backbone of these carotenes [135]. None of the CCD4 characterized so far catalyzes the cleavage of phytoene, in agreement with the hypothesis that a double bond in the carotenoid structure must be adjacent to the cleaved double bond [92,98]. In summary, in vitro and in vivo assays for most of the CCD4 enzymes point to β-carotene and C9–C10 or C90–C100 double bond cleavage as the favorite substrate and positions, respectively, although some relevant exceptions, such as citrus or grape CCD4, have been identified regarding the substrate or cleavage site. In particular, the exclusive cleavage activity reported for citrus CCD4b1 and CCD4, together with its restricted expression pattern, may indicate that this enzyme belongs to a novel class of CCD more functionally related to the recently characterized CCD2 from saffron [117,118]. In planta data, derived from the analysis of apocarotenoid profiles, the alteration in carotenoid complement in ccd4 mutants and overexpressed/repressed CCD4 plants, or both, point to xanthophylls as the preferential substrates in vivo [130,133,134,145]. Nevertheless, in some species and tissues such as potato tubers, chrysanthemum petals, or peach fruit, β-carotene cannot be ruled out as the primary target since alterations in the xanthophylls composition in defective or overexpressing CCD4 plants can be explained by a reduction or increase in β-carotene content as the precursor of β-xanthophylls [136,150,153]. These differences in CCD4 substrate preferences may also reflect the differences in substrate availability or accessibility depending on the type of assay. In the in vitro and in vivo assays, the enzyme has direct access to the potential substrates; this clearly differs from the in planta situation, where carotenoids are integrated in the suborganellar structures of the plastids, and CCD4 is preferentially associated with plastoglobuli, where xanthophylls are synthesized and accumulated. The CCD4 activity on linear carotenes is usually very weak either in vivo or in vitro, with the exception of grapevine CCD4 [98]. Recently, the phenotypic characteristics of the Arabidopsis double mutant clb5 ccd4 (defective in ZDS and CCD4) suggests that, under this particular scenario of hyper-accumulation of linear upstream carotenes, CCD4 generates an apocarotenoid derived from phytofluene, ζ-carotene, or both, indicating an activity on the backbone of these carotenes [135]. None of the CCD4 characterized so far catalyzes the cleavage of phytoene, in agreement with the hypothesis that a double bond in the carotenoid structure must be adjacent to the cleaved double bond [92,98].

4.2.4. Expression Pattern in Plant Tissues CCD4 genes are predominantly expressed in flower and fruit tissues, suggesting specific roles in these organs [130,147]. Usually at least one CCD4 member is highly or exclusively expressed in flowers, and its function has been related to the production of nor-isoprenoid aroma volatiles to attract pollinators [96,133,146,148,154,155]. In chrysantemum, Osmanthus, and cabbage, the expression of a specific CCD4 gene in petals is responsible for carotenoid degradation and a lack of flower coloration [136,145,152]. In chrysanthemum, CCD4a is represented by a small multigene family, and the white to yellow color gradation in petals among different cultivars can be explained by different combinations and levels of expression of specific CCD4a genes [145]. In the flower petals of lily (Lilium brownii var. colchesteri), CCD4 transcript levels are associated with a loss of carotenoid content during anthesis, resulting in flower whitening [156]. In saffron, the expression of CCD4a/b correlates with the production of the nor-isoprenoid β-ionone in flower tissues, while CCD4c is restricted Int. J. Mol. Sci. 2016, 17, 1781 22 of 38 to stigmas and shows a highly regulated expression pattern during flower development [89,130]. In other species, such as sweet orange, tomato, and potato, at least one CCD4 gene is expressed in flowers, but their involvement in carotenoid degradation has not been elucidated [133,148,150]. Expression of CCD4 genes has also been reported in many fruits and its role has been associated with the biosynthesis of nor-isoprenoid volatiles or apocarotenoids pigments, which may attract animals to facilitate seed dispersion. In grape, CCD4a and b genes are expressed in the berry, but only CCD4b shows a fruit-specific expression profile, highly upregulated during ripening, linking its activity to nor-isoprenoid volatiles production [98]. In summer squash (Cucurbita pepo) varieties with white, green, and yellow-orange coloration, the expression of two CCD4 genes (a, b) correlates inversely with carotenoid accumulation in the peel and flesh; therefore, both genes are good candidates for regulating fruit color [111,157]. One of the best-characterized examples of the CCD4 role in fruit pigmentation has been described in peach (Prunus persica). White-fleshed peach cultivars contain at least 10 times less carotenoid concentration and higher nor-isoprenoid carotenoid-derived volatiles than the yellow ones [158]. Recently, several studies have established that alterations in the CCD4 gene are directly related to carotenoid content in yellow peach varieties. In yellow cultivars, CCD4 gene expression is significantly reduced, the formation of a truncated CCD4 protein avoids carotenoids degradation in fruit mesocarp, or both [159–162]. In mandarins and oranges, the specific expression of the CCD4b1/CCD4 gene in the fruit peel during ripening is responsible for the asymmetric cleavage of β-cryptoxanthin and zeaxanthin to generate the C30 apocarotenoids 3-OH-β-8-apocarotenal (β-citraurin) and β-apo-8-apocarotenal [131,133], respectively. It is interesting to note that, due to the intense orange-red color of these C30 apocarotenoids, the citrus CCD4 activity enhances fruit pigmentation, in contrast to the other CCD4s described so far whose activity is associated with color loss. In other species, such as bitter melon, goji berry, and tomato, the expression of at least one CCD4 gene has been reported in ripened fruits, but no relationship has been established with carotenoid content [148,155,163]. In potato tubers, compared with the yellow ones, CCD4 activity regulates coloration, as indicated by the elevated CCD4 expression in mature white-fleshed tubers [150]. Moreover, in CCD4 RNAi potato lines, total carotenoid content was enhanced in tubers (up to 5.6-fold) and flowers, but not in other plant organs [150]. The function of this enzyme in tubers is probably associated with stress response since the potato tubers from RNAi lines showed diverse developmental alterations linked to heat stress phenotypes [150]. Recently, it has been proposed that an apocarotenoid derived from β-carotene in potato tubers could act as a signaling molecule in stress responses [153]. In other plant species, CCD4 expression is also modulated by specific abiotic stresses. Saffron CCD4a and b are expressed in leaf tissues upon dehydration or heat stress as well as in senescence leaves [89]. Moreover, CCD4c is upregulated in flower stigmas subjected to different stress treatments, such as osmotic, wounding, and cold or heat stresses [130]. In B. rapa and B. oleracea seedlings, CCD4 transcript levels increase in response to different abiotic stresses as well as to exogenous treatments with SLs and ABA phytohormones, suggesting their involvement in plant stress resistance response [152]. In other processes involving cellular responses to stress, such as dark-induced leaf senescence or seed maturation and desiccation, a major role of CCD4 in carotenoid degradation has been proposed [107]. These processes are associated with severe alterations in chloroplast structures, causing disassembly of photosystems and light-harvesting complexes and the release of carotenoids, in which CCD4 activity may play a crucial role in modulating turnover of β,β-carotenoids. By contrast, high-light stress in Arabidopsis leaves causes a strong downregulation in CCD4 expression [164], which may prevent cleavage of xanthophylls with a photoprotective function [134]. A role of CCD4 maintaining carotenoid homeostasis has also been reported in Arabidopsis overexpressing PSY [134]. In these plants, CCD4 activity is essential for the formation of apocarotenoid (C13) glycosides (derived from epoxidized xanthophylls) in leaves and long-chain apocarotenoids (derived from β-carotene) in roots. Moreover, a lethal phenotype was observed in PSY-overexpressing seedlings defective in CCD4 activity, supporting the key role of CCD4 as a first step in detoxifying excess of carotenoids Int. J. Mol. Sci. 2016, 17, 1781 23 of 38 into apocarotenoid glycosides [134]. The CCD4 activity is also involved in the formation of an uncharacterized apocarotenoid, most likely derived from ζ-carotene, phytofluene, or both, participating in a novel signaling process that regulates early chloroplasts and leaf development [135]. In summary, the presence of different CCD4 members with tissue- and organ-specific expression patterns in many plant species indicates a high functional diversification within this subfamily. In the last few years, we have gained much knowledge on the physiological, biochemical, and molecular regulation of CCD4 enzymes and their presumed involvements in different processes. In one case, the function is concerned with the production of apocarotenoids in flowers and fruits to attract pollinators and seed dispersers, which has eco-physiological and reproductive implications. In another case, the role is directly involved in carotenoid turnover, related to specific stress conditions and alterations of the carotenoid pathway. A third function was recently identified with the involvement of CCD4 activity in the formation of an apocarotenoid-derived signal that regulates early chloroplasts and leaf development. Future research on different CCD4 members in multigene families, including structural, functional, and spatial studies as well potential interactions with other enzymes of the carotenoid pathway, will help towards a better understanding as to how carotenoid content is modulated in different plant organs.

4.3. The CCD7 and CCD8 Subfamilies The first report on CCD7 and CCD8 enzymes in plants derives from the max4 mutant of Arabidopsis, affected in the AtCCD8 gene (Figure1). The mutants exhibit an increase in lateral branching, which reminiscent of the phenotype of the max3 mutant, which shows an increase in lateral branching as a result of a mutation in the AtCCD7 gene, indicating that both AtCCD8 and AtCCD7 are involved in this developmental process [165,166]. The biochemical characterization of AtCCD7 and AtCCD8 [167] demonstrated that AtCCD7 catalyzes a C9–C10 cleavage of β-carotene to produce 0 10 -apo-β-carotenal (C27) and β-ionone (C13). The AtCCD8 protein is able to catalyze a secondary 0 cleavage of 10 -apo-β-carotenal at the C13–C14 position to produce 13-apo-β-carotenone (C18). However, it has been shown that CCD7 is also active on many other carotenoid substrates [166,167]. In contrast to CCD1, which has a cytoplasmatic location, CCD7 and CCD8 are localized in plastids, as CCD4 and CCD2 are, the predominant sites for carotenoid accumulation [87]. The CCD7 and CCD8 enzymes are involved in the biosynthesis of a relatively novel class of apocarotenoid hormones, the strigolactones (SLs), which control lateral shoot growth and which appear to be well conserved among the studied plant species. Branching mutants from Arabidopsis, petunia, pea, and rice lacking CCD7 or CCD8 have reduced SL concentrations, and applications of synthetic SLs to the mutant restores the wild-type branching phenotype [87,168,169]. In tomato, a reduction of CCD7 expression increases branching [170]. This is the case of the tomato mutant Sl-ORT1, which is deficient in SLs and has reduced CCD7 expression [171]. These enzymes have been identified in all high plant genomes sequenced up to date. SLs are derived from carotenoids, thus belonging to the class of the apocarotenoids that function as signaling molecules. This is exemplified by their role promoting germination of the parasitic plants Striga and Orobanche [172] or their involvement in symbiotic interaction with arbuscular mycorrhizal fungi [173]. CCD7 and CCD8 are involved in sequential cleavage reactions needed for the synthesis of SLs. These metabolites share a common C19 structure consisting of a tricyclic lactone connected via an enol ether bridge to a second lactone (Figure4). The first steps of SL synthesis involve isomerization and dioxygenase-mediated cleavage of a carotenoid precursor via plastidic isomerase action by D27, followed by the cleavage activity of CCD7 and CCD8. MAX1 encodes a cytP450 enzyme predicted to act downstream of CCD7 and CCD8, and is required for the synthesis of active SLs. SL signaling requires the hormone-dependent interaction of the α/β hydrolase protein DWARF 14 (D14), a likely SL receptor, with DWARF 3 (D3), an F-box component of the Skp-Cullin-F-box (SCF) E3 ubiquitin ligase complex. The third component in the signaling pathway is D53, which shares predicted features with the class I Clp ATPase proteins and can form a complex with D14 and D3. SLs induce D53 degradation by the proteasome and abrogate its Int. J. Mol. Sci. 2016, 17, 1781 24 of 38 activity in promoting axillary bud outgrowth [174]. Analysis of mutants, such as D27, D17/CCD7, and D10/CCD8 from rice, showed that all these genes are involved in the biosynthesis of SLs. However, mutantsInt. J. Mol. Sci. lacking 2016, 17 one, 1781 or more of these genes are able to produce SL metabolites, which indicate24 of an 37 alternative minor pathway for SL biosynthesis [175]. The firstfirst identifiedidentified function of SLs in higherhigher plantsplants was as aa stimulatorystimulatory signal in seedseed germination ofof rootroot parasitic parasitic weeds, weeds, such such as as the the witchweed witchweedStriga Strigaspp. spp. and and broomrapes broomrapes (Orobanche (Orobancheand Phelipancheand Phelipanchespp.) spp.) [176]. [176]. The first The germination first germination stimulant stimulant identified identified for Striga forwas Striga called was strigol, called a strigol, member a ofmember SLs isolated of SLs from isolated cotton from (Gossypium cotton hirsutum (GossypiumL.) root hirsutum exudates L.) [177root]. Manyexudates other [177]. compounds Many other with similarcompounds structures with similar and with structures germination and with stimulatory germination properties stimulatory were discoveredproperties were in root discovered exudates ofin hostroot plantexudates species of [178host]. Orobancheplant speciesand Phelipanche [178]. Orobanchespp. are and obligate Phelipanche non-photosynthetic spp. are obligate holoparasites non- thatphotosynthetic depend fully holoparasites on their host that for depend nutritional fully needs. on thei Itr ishost not for clear nutritional how these needs. parasites It is not perceive clear how SLs. Recently,these parasites it has perceive been suggested SLs. Recently, that MAX2, it has been AtD14, suggested or both, that which MAX2, are sensitiveAtD14, or to both, SLs, which might are be involvedsensitive to in SLs, this processmight be [179 involved]. In rice, in this the process obstruction [179]. of In the rice, carotenoid the obstruction biosynthesis of the in carotenoid different stepsbiosynthesis of the pathway, in different including steps of those the leadingpathway, to CCD7including substrates, those lead leding to reduced to CCD7 SLs substrates, production led and to secretionreduced SLs into production the rhizosphere, and secretion resulting into in decreased the rhizosphere,Striga germination resulting in and decreased consequently Striga germination lower Striga infectionand consequently [180]. lower Striga infection [180].

Figure 4. Biosynthetic pathway of strigolactones (SLs) from β-carotene. Figure 4. Biosynthetic pathway of strigolactones (SLs) from β-carotene.

The analysis of genomes from several plant species showed a single copy for the CCD7 gene. The analysis of genomes from several plant species showed a single copy for the CCD7 gene. However, in most of the analyzed genomes, CCD8 is present as several copies [144]. In some cases, However, in most of the analyzed genomes, CCD8 is present as several copies [144]. In some cases, as in apple (M. domestica) and camelina (Camelina sativa), CCD8 genes are organized in tandem in the as in apple (M. domestica) and camelina (Camelina sativa), CCD8 genes are organized in tandem in same chromosome (Tables S2 and S3). CCD7 and CCD8, as other members of CCDs genes, contain the same chromosome (Tables S2 and S3). CCD7 and CCD8, as other members of CCDs genes, introns, with the exception of the NCED related group, which is intron-less. The CCD7 genes are contain introns, with the exception of the NCED related group, which is intron-less. The CCD7 characterized by the presence of 4–7 introns, four of which are well conserved among different genes are characterized by the presence of 4–7 introns, four of which are well conserved among plant species. The number of introns in CCD8 sequences ranges between 2 and 6, with 5 being the most frequent number among the species investigated. The identities among the CCD7 and CCD8 paralogous genes range between 36% and 100% and between 45% and 100%, respectively. The phylogenetic analysis of CCD7 proteins shows three clusters distinguishing the Embryophyte, Grass, and Eudicot phyla. However, a similar analysis with CCD8 proteins did not so clearly match this taxonomical classification (Figure S3). Int. J. Mol. Sci. 2016, 17, 1781 25 of 38 different plant species. The number of introns in CCD8 sequences ranges between 2 and 6, with 5 being the most frequent number among the species investigated. The identities among the CCD7 and CCD8 paralogous genes range between 36% and 100% and between 45% and 100%, respectively. The phylogenetic analysis of CCD7 proteins shows three clusters distinguishing the Embryophyte, Grass, and Eudicot phyla. However, a similar analysis with CCD8 proteins did not so clearly match this taxonomical classification (Figure S3). In contrast to the high functional conservation of the CCD7 and CCD8 enzymes, their genes present different expression patterns. Previous works in some plants, e.g., in rice, indicated that CCD7 and CCD8 genes are found to be upregulated in root [181]. In the columella cap of primary and lateral roots, CCD8 is highly expressed [182,183], and exogenous treatment with auxin 1-naphthaleneacetic acid (NAA) led to an overexpression of CCD8 expression in the primary root and cortical tissue. The expression of CCD8 in roots tissues from Arabidopsis [87], petunia [184], pea [185], kiwi [186], and tomato [187] exceeds shoot expression. However, in saffron and chrysanthemum, the opposite behavior was detected [188]; in rose, no transcripts are found in roots [189]. High expression of CCD7 in root from Arabidopsis is observed, [166], but more recently even higher transcripts were observed in seeds and in the stem vascular tissue [190]. Different patterns of CCD7 expression was found in rice [191], petunia [192], and tomato [170]. High expression was detected in vascular bundle tissue in rice, while CCD7 was mainly expressed in internodes and nodes in petunia, and CCD7 expression was low in green tissue in tomato in comparison to root and shoots. These differences suggest different mechanisms of the SL-regulation of the shoot branching of the root and shoot in different species. In addition to regulating shoot architecture and branching, SLs are involved in many other processes, including root growth, root hair elongation, lateral root formation, adventitious rooting, stem elongation, leaf expansion, leaf senescence, secondary growth, and drought and salinity responses [183,193–196]. CCD7 and CCD8 from soybean are involved in abiotic stress physiology, and their expression levels were greatly influenced by exogenous ABA treatment [112]. CCD7 from Lotus japonicus affects reproduction by reducing the number of flowers, fruits, and seeds and modulates leaf senescence and abscission [197]. CCD8 from kiwifruit modifies branch development and slows up leaf senescence [186]. An analogous association was detected in the rice tillering dwarf mutant D3, and the mutant MAX2/ORE9 from Arabidopsis [198]. Nevertheless, the delayed leaf senescence was not observed in the rms4 mutant from pea [199], suggesting that the connection between leaf senescence and branch development has followed a convergent evolution in plant species. Recent experimental evidence has revealed that auxin influences pea mycorrhizal symbiosis by regulating the production of SL plant hormones. During the symbiosis between the auxin deficient bsh mutant and the mycorrhizal fungus Glomus intraradices, the low auxin production correlated with reduced mycorrhizal colonization, SL levels, and biosynthetic gene expression, including CCD7 and CCD8 from pea [200]. In Crocus sativus, CCD7 and CCD8 were identified in saffron corms and stigmas, leading to attribute novel roles to SLs in this plant [201]. In corms, SLs act synergically with auxin to arrest the outgrowth of the axillary buds. In stigma tissue, transcripts were detected at a higher level than in vascular tissues, leaves, and roots. The abundance of both transcripts in immature orange stigmas suggests that these enzymes and SLs might regulate procambial activity and the development of new vascular tissues connecting leaves with the mother corm. A similar function was already suggested for SLs in flower development in petunia [184]. There has been great progress over the past years in characterizing plant CCD enzymes and their apocarotenoid products. As more functions are unraveled for apocarotenoids in plants, more additional roles will be assigned to CCD7 and CCD8. Further characterization of these genes may provide hints about the origin of parasitism, as well as new approaches for controlling parasitic plants and understanding important physiological processes of the underlying metabolic pathways. Int. J. Mol. Sci. 2016, 17, 1781 26 of 38

5. CCDs in Algae Algae are classified throughout many divisions of the kingdom Plantae. Their sizes range from single cells of picophytoplankton to seaweeds. Algae can synthesize many kinds of carotenoids that are absent in higher plants and therefore have been proposed as excellent chemotaxonomic markers [202,203]. Only few studies have been conducted with CCDs in algae. The best-known apocarotenoid in algae is retinal [204,205]. Most unicellular flagellate algae are phototactic [206]. They have developed an eyespot, which have been most studied in Chlamydomonas reinhardtii. The eye of Chlamydomonas comprises the optical system and at least five different rhodopsin photoreceptors. Two of these receptors are rhodopsin-ion channel hybrids switching between closed and open states by photoisomerization of an attached retinal chromophore [207]. The proteome of the eyespot apparatus in C. reinhardtii includes putative carotenoid cleavage dioxygenases homologous to Synechocystis sp. PCC 6803 ACOX_SYNY3 [208], which forms retinal from diverse apo-carotenoids in vitro [29]. However, the phylogenetic analysis of the CCD sequences present in the databases from algae did not show the presence of such homologues; instead, only CCD7 and CCD8 homologues have been identified (Table S2, Figure S4). As mentioned in the former section, SLs comprise an important class of apocarotenoid derivatives acting as hormones in plants [209] and have also been found in several algae [210]. A recent study showed that freshwater green algae belonging to the Charales, some of the closest freshwater green algal relatives of land plants, produce and exude SLs. The same study showed the presence of CCD7 and CCD8 homologues in different green algae taxa (Table S2, Figure S4) [211]. The authors suggested that SLs could play a role in rhizoid elongation in algae and thus increase their anchorage ability. Other important apocarotenoids in algae are the carotenoid-derived volatiles, which are released by diverse algae taxa and influence aquatic odors [212]. Ulva prolifera, Ulva linza, Monostroma nitidum, Ulothrix fimbriata, and Porphyra tenera produce volatile apocarotenoids in a high proportion. These C13 apocarotenoids or their derivatives exhibit growth-regulating properties in algae [213]. In addition, they may play ecological roles in providing competitive advantages, e.g., by inhibiting the growth of surrounding phytoplankton [214]. Functional carotenoid cleavage-like enzymes are expected to contribute to the formation of volatile apocarotenoids in these macro-algae [215]. No specific CCD has been isolated and identified as responsible for the production of these apocarotenoids, but the presence of homologous sequences closely related to CCD1 and CCD4 subfamilies (Table S2, Figure S4) suggests that theses CCDs could be good candidates to mediate the formation of these apocarotenoids volatiles.

6. Conclusions In nearly 20 years, the research on CCDs enzymes has evolved quickly, with the cloning of many different CCDs from plants and microorganisms. The increasing number of biochemical analyses for newly identified CCDs evidences the functional diversification of these enzymes in different species, adapted to a diversity of substrates and cleaving sites in a large variety of organisms. More recently, the knowledge on new putative CCDs is increasing enormously with the breakthrough of genome sequencing projects and transcriptome analyses of many additional species. The experimental basis for their future characterization has been solidly established, and the functional diversity found so far allows anticipating that new carotenoid or apocarotenoid substrates for novel CCD enzymatic reactions will be discovered in the next years. The biological purposes of such reactions will be predictably more elusive. Although the function of several novel CCDs has been resolved by in vitro analysis, we are far from understanding the activities and function of these enzymes in vivo in the different organisms, and more efforts will be needed to learn about how these CCDs are regulated at metabolic, transcriptional, and translational levels. The functions for newly identified CCDs and apocarotenoid products will be a future challenge, and effective research will require integrated multidisciplinary approaches to advance in this fascinating field of research.

Supplementary Materials: Supplementary materials can be found at www.mdpi.com/1422-0067/17/11/1781/s1. Int. J. Mol. Sci. 2016, 17, 1781 27 of 38

Acknowledgments: This work was supported by the Ibero-American network for the study of carotenoids as food ingredients (IBERCAROT, CYTED 112RT0445), the Spanish Government, Ministry of Science and Technology (projects AGL2015-70218, BIO2013-44239-R, AGL2012-34573, BIO2012-39716, and BIO2009-11131), the Andalusian Government (projects P07-CVI-02813 and CTS-6638), and the Generalitat Valenciana (PROMETEOII/2014/027). Author Contributions: All authors reviewed the literature and wrote this manuscript. Conflicts of Interest: The authors declare no conflict of interest.

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