Carotenoid Cleavage Oxygenases Research Articles

A nuclear receptor heterodimer, CgPPAR2-CgRXR, acts as a regulator of carotenoid metabolism in Crassostrea gigas

Nuclear receptors (NRs) are mostly ligand-activated transcription factors in animals and play essential roles in metabolism and homeostasis. The NR heterodimer composed of PPAR/RXR (peroxisome proliferator-activated receptor/retinoidXreceptor) is considered a key regulator of lipid metabolism in vertebrate. However, in molluscs, how this heterodimer is involved in carotenoid metabolism remains unclear. To elucidate how this heterodimer regulates carotenoid metabolism, we identified a PPAR gene in C. gigas, designated as CgPPAR2 (LOC105323212), and functionally characterized it using two-hybrid and reporter systems. CgPPAR2 is a direct orthologue of vertebrate PPARs and the second PPAR gene identified in C. gigas genome in addition to CgPPAR1 (LOC105317849). The results demonstrated that CgPPAR2 protein can form heterodimer with C. gigas RXR (CgRXR), and then regulate carotenoid metabolism by controlling carotenoid cleavage oxygenases with different carotenoid cleavage efficiencies. This regulation can be affected by retinoid ligands, i.e., carotenoid derivatives, validating a negative feedback regulation mechanism of carotenoid cleavage for retinoid production. Besides, organotins may disrupt this regulatory process through the mediation of CgPPAR2/CgRXR heterodimer. This is the first report of PPAR/RXR heterodimer regulating carotenoid metabolism in mollusks, contributing to a better understanding of the evolution and conservation of this nuclear receptor heterodimer.

Charge Maintenance during Catalysis in Nonheme Iron Oxygenases.

Here, the choice of the first coordination shell of the metal center is analyzed from the perspective of charge maintenance in a binary enzyme-substrate complex and an O2-bound ternary complex in the nonheme iron oxygenases. Comparing homogentisate 1,2-dioxygenase and gentisate dioxygenase highlights the significance of charge maintenance after substrate binding as an important factor that drives the reaction coordinate. We then extend the charge analysis to several common types of nonheme iron oxygenases containing either a 2-His-1-carboxylate facial triad or a 3-His or 4-His ligand motif, including extradiol and intradiol ring-cleavage dioxygenases, thiol dioxygenases, α-ketoglutarate-dependent oxygenases, and carotenoid cleavage oxygenases. After forming the productive enzyme-substrate complex, the overall charge of the iron complex at the 0, +1, or +2 state is maintained in the remaining catalytic steps. Hence, maintaining a constant charge is crucial to promote the reaction of the iron center beginning from the formation of the Michaelis or ternary complex. The charge compensation to the iron ion is tuned not only by protein-derived carboxylate ligands but also by substrates. Overall, these analyses indicate that charge maintenance at the iron center is significant when all the necessary components form a productive complex. This charge maintenance concept may apply to most oxygen-activating metalloenzymes systems that do not draw electrons and protons step-by-step from a separate reactant, such as NADH, via a reductase. The charge maintenance perception may also be useful in proposing catalytic pathways or designing prototypical reactions using artificial or engineered enzymes for biotechnological applications.

Evolutionary Origin of the Carotenoid Cleavage Oxygenase Family in Plants and Expression of Pepper Genes in Response to Abiotic Stresses.

Plant carotenoid cleavage oxygenase (CCO) is an enzyme that catalyzes the synthesis of carotenoids and participates in many important physiological functions. The plant CCOs exist in two forms, namely carotenoid cleavage dioxygenase (CCD) and nine-cis epoxide carotenoid dioxygenase (NCED). Although studies have shown that this gene family has been identified in many species, such as Arabidopsis, grape, and tomato, the evolutionary origin of the CCO family and the expression pattern of pepper genes in response to H2O2 and other abiotic stresses are still unclear. In this study, we used the bioinformatics method to identify and analyze the members of the CCO gene family from pepper and other 13 plants from lower to higher plant species based on the whole genome sequence. A total of 158 CCO genes were identified in different plant species and further divided into two groups (e.g., groups I and II). The former was subdivided into CCD7 and CCD8 and have independent evolutionary origins, respectively, while the latter was subdivided into CCD1, CCD4, CCD-like, and NCED, which may have come from a common ancestor. In addition, the results of RNA-seq showed that the expression patterns of pepper CaCCO genes were different in the tissues tested, and only few genes were expressed at high levels such as CaCCD1a, CaCCD4a, CaNCED3, and CaCCD1b. For hydrogen peroxide (H2O2) and other abiotic stresses, such as plant hormones, heat, cold, drought, and NaCl treatments, induction of about half of the CaCCO genes was observed. Moreover, the expression patterns of CaCCOs were further investigated under heat, cold, drought, and NaCl treatments using quantitative real-time PCR (qRT-PCR), and most members were responsive to these stresses, especially some CaCCOs with significant expression changes were identified, such as CaCCD4c, CaCCD-like1, CaCCD8, and CaCCD1b, suggesting the important roles of CaCCOs in abiotic stress responses. All these results will provide a valuable analytical basis for understanding the evolution and functions of the CCO family in plants.

Vitamin A Transporters in Visual Function: A Mini Review on Membrane Receptors for Dietary Vitamin A Uptake, Storage, and Transport to the Eye.

Vitamins are essential compounds obtained through diet that are necessary for normal development and function in an organism. One of the most important vitamins for human physiology is vitamin A, a group of retinoid compounds and carotenoids, which generally function as a mediator for cell growth, differentiation, immunity, and embryonic development, as well as serving as a key component in the phototransduction cycle in the vertebrate retina. For humans, vitamin A is obtained through the diet, where provitamin A carotenoids such as β-carotene from plants or preformed vitamin A such as retinyl esters from animal sources are absorbed into the body via the small intestine and converted into all-trans retinol within the intestinal enterocytes. Specifically, once absorbed, carotenoids are cleaved by carotenoid cleavage oxygenases (CCOs), such as Beta-carotene 15,15’-monooxygenase (BCO1), to produce all-trans retinal that subsequently gets converted into all-trans retinol. CRBP2 bound retinol is then converted into retinyl esters (REs) by the enzyme lecithin retinol acyltransferase (LRAT) in the endoplasmic reticulum, which is then packaged into chylomicrons and sent into the bloodstream for storage in hepatic stellate cells in the liver or for functional use in peripheral tissues such as the retina. All-trans retinol also travels through the bloodstream bound to retinol binding protein 4 (RBP4), where it enters cells with the assistance of the transmembrane transporters, stimulated by retinoic acid 6 (STRA6) in peripheral tissues or retinol binding protein 4 receptor 2 (RBPR2) in systemic tissues (e.g., in the retina and the liver, respectively). Much is known about the intake, metabolism, storage, and function of vitamin A compounds, especially with regard to its impact on eye development and visual function in the retinoid cycle. However, there is much to learn about the role of vitamin A as a transcription factor in development and cell growth, as well as how peripheral cells signal hepatocytes to secrete all-trans retinol into the blood for peripheral cell use. This article aims to review literature regarding the major known pathways of vitamin A intake from dietary sources into hepatocytes, vitamin A excretion by hepatocytes, as well as vitamin A usage within the retinoid cycle in the RPE and retina to provide insight on future directions of novel membrane transporters for vitamin A in retinal cell physiology and visual function.

SlCCD1A Enhances the Aroma Quality of Tomato Fruits by Promoting the Synthesis of Carotenoid-Derived Volatiles.

The loss of volatiles results in the deterioration of flavor in tomatoes. Volatiles are mainly derived from fatty acid, carotenoid, phenylpropane, and branched chain amino acids. In this study, the tomato accession CI1005 with a strong odor and accession TI4001 with a weak odor were analyzed. The volatile contents were measured in tomato fruits using gas chromatography-mass spectrometry. The scores of tomato taste and odor characteristics were evaluated according to hedonistic taste and olfaction. It was found that the content of fatty acid-derived volatiles accounted for more than half of the total volatiles that had grassy and fatty aromas. Phenylpropane-derived volatiles had irritation and floral aromas. Branched-chain amino acid-derived volatiles had a caramel aroma. Carotenoid-derived volatiles had floral, fruity, fatty, and sweet-like aromas, preferred by consumers. A lack of carotenoid-derived volatiles affected the flavor quality of tomato fruits. The accumulation of carotenoid-derived volatiles is regulated by carotenoid cleavage oxygenases (CCDs). A tissue-specific expression analysis of the SlCCD genes revealed that the expression levels of SlCCD1A and SlCCD1B were higher in tomato fruits than in other tissues. The expression levels of SlCCD1A and SlCCD1B were consistent with the trend of the carotenoid-derived volatile contents. The expression of SlCCD1A was higher than that for SlCCD1B. A bioinformatics analysis revealed that SlCCD1A was more closely linked to carotenoid metabolism than SlCCD1B. The overexpression of SlCCD1A indicated that it could cleave lycopene, α-carotene, and β-carotene to produce 6-methyl-5-hepten-2-one, geranylacetone, α-ionone, and β-ionone, increasing the floral, fruity, fatty, and sweet-like aromas of tomato fruits. The flavor quality of tomato fruits could be improved by overexpressing SlCCD1A.

Genome-wide characterization of carotenoid oxygenase gene family in three cotton species and functional identification of GaNCED3 in drought and salt stress.

Cotton that serves natural fiber for the textile industry is an important industrial crop. However, abiotic stress imposed a significant negative impact on yield and quality of cotton fiber. Carotenoid cleavage oxygenases (CCOs) that specifically catalyze the cleavage of carotenoid are essential for plant growth and development and abiotic stress response. While information of cotton CCOs and their potential functions in abiotic stress is still far from satisfactory, which imposes restrictions on application in genetic breeding for stress resistance. In this study, 15, 15, and 30 CCOs were identified from Gossypium arboreum, Gossypium raimondii, and Gossypium hirsutum, respectively. Phylogenetic relationship indicated that CCO genes could be classified into two groups (NCEDs and CCDs). Cis-elements prediction showed that there were 18 types of stress-related cis-elements in promoter regions. Analysis with transcriptome data revealed tissue-specific expression pattern of cotton CCOs. qRT-PCR analysis revealed only that GhNCED3a_A/D and GhNCED3c_A/D had strong response to drought, salt, and cold stress, while GhCCD1_A/D and GhCCD4_A showed relatively slight expression changes. Virus-induced gene silencing of GaNCED3a, the ortholog gene of GhNCED3a_A/D, suggested that silenced plants exhibited decreased resistance not only to drought but also to salt, with significantly reduced proline content, and high malondialdehyde content and water loss rate. In addition, stress response genes RD29A, DREB1A, and SOS1 significantly downregulated under drought and salt stress in silenced plants compared to control plants, indicating that GaNCED3a played an important role in drought and salt response. The results provided valuable insights into function analysis of cotton CCOs in abiotic stress response, and suggested potential benefit genes for stress-resistant breeding.

Genome-Wide Identification and Expression Analysis of the Carotenoid Cleavage Oxygenase Gene Family in Five Rosaceae Species

The carotenoid cleavage oxygenases (CCOs), which include carotenoid cleavage dioxygenases (CCDs) and 9-cis-epoxycarotenoid dioxygenases (NCEDs), are enzymes that are critical to the mediation of the degradation of carotenoids to apocarotenoids. In this study, we identified a total of 12, 20, 11, 8, and 10 CCO genes from Pyrus bretschneideri, Malus domestica, Fragaria vesca, Prunus mume, and Prunus persica, respectively. Phylogenetic analysis showed that these CCO genes clustered into six groups (NCED, CCD1, CCD4, CCD7, CCD8, and CCD-like). Gene structure analysis revealed that CCO genes within the same groups showed similar exon numbers and contained similar motifs. Duplication analysis revealed that tandem duplication played a significant role in the expansion of CCO members in Malus domestica. Purifying selection was involved in the evolution of CCO genes in Pyrus bretschneideri. The transcriptomic data-based expression analysis revealed that the CCO genes exhibited distinct patterns of expression in the roots, leaves, buds, and fruit. The expression patterns based on qRT-PCR showed that there were greater differences on the relative expression levels of PbCCD1 and PbCCD5 between cultivars “Enli” (inflorescence sparsely branched) and “Jinxiangshui” (inflorescence copiously branched) in the three different stages of flower bud morphological differentiation, revealing the possibility of involvement of these genes in the branching of the pear inflorescence. This study provides a valuable resource for further investigations of CCO gene functions in Rosaceae species.

Genome-wide identification, characterization and expression analysis of the carotenoid cleavage oxygenase (CCO) gene family in Saccharum

Carotenoid cleavage oxygenases (CCOs) play crucial roles in plant growth and development, as well as in the response to phytohormonal, biotic and abiotic stresses. However, comprehensive and systematic research on the CCO gene family has not yet been conducted in Saccharum. In this study, 47 SsCCO and 14 ShCCO genes were identified and characterized in Saccharum spontaneum and Saccharum spp. R570 cultivar, respectively. The SsCCOs consisted of 38 SsCCDs and 9 SsNCEDs, while ShCCOs contained 11 ShCCDs and 3 ShNCEDs. The SsCCO family could be divided into 7 groups, while ShCCO family into 5 groups. The genes/proteins contained similar compositions within the same group, and the evolutionary mechanisms differed between S. spontaneum and R570. Gene Ontology annotation implied that CCOs were involved in many physiological and biochemical processes. Additionally, 41 SsCCOs were regulated by 19 miRNA families, and 8 ShCCOs by 9 miRNA families. Cis-regulatory elements analysis suggested that CCO genes functioned in the process of growth and development or under the phytohormonal, biotic and abiotic stresses. qRT-PCR analysis indicated that nine CCO genes from different groups exhibited similar expression patterns under abscisic acid treatment, while more divergent profiles were observed in response to Sporisorium scitamineum and cold stresses. Herein, comparative genomics analysis of the CCO gene family between S. spontaneum and R570 was conducted to investigate its evolution and functions. This is the first report on the CCO gene family in S. spontaneum and R570, thus providing valuable information and facilitating further investigation into its function in the future.

The Structural and Biochemical Basis of Apocarotenoid Processing by β-Carotene Oxygenase-2.

In mammals, carotenoids are converted by two carotenoid cleavage oxygenases into apocarotenoids, including vitamin A. Although knowledge about β-carotene oxygenase-1 (BCO1) and vitamin A metabolism has tremendously increased, the function of β-carotene oxygenase-2 (BCO2) remains less well-defined. We here studied the role of BCO2 in the metabolism of long chain β-apocarotenoids, which recently emerged as putative regulatory molecules in mammalian biology. We showed that recombinant murine BCO2 converted the alcohol, aldehyde, and carboxylic acid of a β-apocarotenoid substrate by oxidative cleavage at position C9,C10 into a β-ionone and a diapocarotenoid product. Chain length variation (C20 to C40) and ionone ring site modifications of the apocarotenoid substrate did not impede catalytic activity or alter the regioselectivity of the double bond cleavage by BCO2. Isotope labeling experiments revealed that the double bond cleavage of an apocarotenoid followed a dioxygenase reaction mechanism. Structural modeling and site directed mutagenesis identified amino acid residues in the substrate tunnel of BCO2 that are critical for apocarotenoid binding and catalytic processing. Mice deficient for BCO2 accumulated apocarotenoids in their livers, indicating that the enzyme engages in apocarotenoid metabolism. Together, our study provides novel structural and functional insights into BCO2 catalysis and establishes the enzyme as a key component of apocarotenoid homeostasis in mice.

Research Advances on Biosynthesis, Regulation, and Biological Activities of Apocarotenoid Aroma in Horticultural Plants

Apocarotenoids, which play important roles in the growth and development of horticultural plants, are produced by the action of carotenoid cleavage oxygenase (CCO) family members or nonenzymatic cleavage actions. Apocarotenoids are commonly found in leaves, flowers, and fruits of many horticultural plants and participate in the formation of pigments, flavors, hormones, and signaling compounds. Some of them are recognized as important aroma components of fruit and flower with aromatic odor, such as βß-ionone, β-damascenone, and 6-methyl-5-hepten-2-one in tomato fruit, and have low odor thresholds with β-ionone having odor threshold of only 0.007 ppb. In this review, the main apocarotenoid aroma components in horticultural plants were listed, and factors influencing their production were discussed at first. Then, the biosynthetic pathway of apocarotenoid aromas was briefly introduced, and the CCDs gene family was highlighted, and the nonenzymatic production of apocarotenoid aromas was also mentioned. Next, chemical and molecular regulations of apocarotenoid aromas and their biological activities were summarized. Finally, further exploration aspects needed were suggested. We anticipate that this review can afford some crucial information for comprehensive application of apocarotenoid volatile compounds in horticultural plants.

Palmitoylation of Metazoan Carotenoid Oxygenases.

Abundant in nature, carotenoids are a class of fat-soluble pigments with a polyene tetraterpenoid structure. They possess antioxidant properties and their consumption leads to certain health benefits in humans. Carotenoid cleavage oxygenases (CCOs) are a superfamily of enzymes which oxidatively cleave carotenoids and they are present in all kingdoms of life. Complexity of CCO evolution is high. For example, in this study we serendipitously found a new family of eukaryotic CCOs, the apocarotenoid oxygenase-like (ACOL) family. This family has several members in animal genomes and lacks the animal-specific amino acid motif PDPCK. This motif is likely to be associated with palmitoylation of some animal CCOs. We recently demonstrated that two mammalian members of the carotenoid oxygenase family retinal pigment epithelial-specific 65 kDa protein (RPE65) and beta-carotene oxygenase 2 (BCO2) are palmitoylated proteins. Here we used the acyl-resin-assisted capture (acyl-RAC) method to demonstrate protein palmitoylation and immunochemistry to localize mouse BCO2 (mBCO2) in COS7 cell line in the absence and presence of its substrate β-carotene. We demonstrate that mBCO2 palmitoylation depends on the evolutionarily conserved motif PDPCK and that metazoan family members lacking the motif (Lancelet beta-carotene oxygenase-like protein (BCOL) and Acropora ACOL) are not palmitoylated. Additionally, we observed that the palmitoylation status of mBCO2 and its membrane association depend on the presence of its substrate β-carotene. Based on our results we conclude that most metazoan carotenoid oxygenases retain the evolutionarily conserved palmitoylation PDPCK motif to target proteins to internal membranes depending on substrate status. Exceptions are in the secreted BCOL subfamily and the strictly cytosolic ancient ACOL subfamily of carotenoid oxygenases.

Plant carotenoid cleavage oxygenases: structure-function relationships and role in development and metabolism.

A plant communicates within itself and with the outside world by deploying an array of agents that include several attractants by virtue of their color and smell. In this category, the contribution of 'carotenoids and apocarotenoids' is very significant. Apocarotenoids, the carotenoid-derived compounds, show wide representation among organisms. Their biosynthesis occurs by oxidative cleavage of carotenoids, a high-value reaction, mediated by carotenoid cleavage oxygenases or carotenoid cleavage dioxygenases (CCDs)-a family of non-heme iron enzymes. Structurally, this protein family displays wide diversity but is limited in its distribution among plants. Functionally, this protein family has been recognized to offer a role in phytohormones, volatiles and signal production. Further, their wide presence and clade-specific functional disparity demands a comprehensive account. This review focuses on the critical assessment of CCDs of higher plants, describing recent progress in their functional aspects and regulatory mechanisms, domain architecture, classification and localization. The work also highlights the relevant discussion for further exploration of this multi-prospective protein family for the betterment of its functional understanding and improvement of crops.

Determination of In Vitro and In Vivo Activities of Plant Carotenoid Cleavage Oxygenases.

Carotenoid cleavage products, apocarotenoids, are biologically active compounds exerting important functions as chromophore, hormones, signaling molecules, volatiles, and pigments. Apocarotenoids are generally synthesized by the carotenoid cleavage dioxygenases (CCDs) that comprise a ubiquitous family of enzymes. The activity of plant CCDs was unraveled more than 20years ago, with the characterization of the maize VP14, the first identified CCD. The protocol developed to determine the activity of this enzyme in vitro is still being used, with minor modifications. In addition, in vivo procedures have been developed during these years, mainly based on the exploitation of Escherichia coli cells engineered to produce specific carotenoid substrates. Further, technological developments have led to significant improvements, contributing to a more efficient detection of the reaction products. This chapter provides an updated set of detailed protocols suitable for the in vitro and in vivo characterization of the activities of CCDs, starting from well-established methods.

Exploring the codon patterns between CCD and NCED genes among different plant species

Plant carotenoid cleavage oxygenase (CCO) is an enzyme which catalyzes carotenoids to apocarotenoid products that are involved in several vital physiological functions. The CCO exists in two forms, namely, CCD (Carotenoid Cleavage Dioxygenase) and NCED (Nine-Cis Epoxycarotenoid Dioxygenase). This paper relates to a comparative study on CCD and NCED genes through phylogeny and codon usage analysis. The result of the phylogenetic analysis indicates a closer relationship between CCD and NCED subclass genes, while the RSCU values indicate a high preference for CUC codon in both CCD and NCED gene families. The mean ENc value of NCED genes was found to be 48.76, suggesting a higher codon bias compared to CCD genes. However, the ENc-GC3S plot suggests that both the gene families are under mutational pressure with variations according to their species-specific role. Similarly, the multivariate analysis also suggests that nucleotide mutation bias influences codon usage. Correlation analysis of Axis I and codon adaptation index values indicate a significant correlation between critical indices. Even though the prominence of the variations in codon usage between the two gene families, they are exerted towards the time-specific functional requirement for that plant species. This is evident from the cleaving roles of these enzymes against various carotenoids at different growth stages. The result of this investigation indicates that CCD and NCED genes are under mutational pressure. This codon bias study paves the way for increasing the production of apocarotenoids, which have a great significance in the industry.

Expression and Characterization of Carotenoid Cleavage Oxygenases From Herbaspirillum seropedicae and Rhodobacteraceae bacterium Capable of Biotransforming Isoeugenol and 4-Vinylguaiacol to Vanillin.

HsCCO and RbCCO from Herbaspirillum seropedicae and Rhodobacteraceae bacterium were selected and characterized from five putative bacterial carotenoid cleavage oxygenase gene sequences, due to merits in expression solubility and catalytic properties. Both enzymes can convert 4-vinylguaiacol and isoeugenol to vanillin. HsCCO showed maximum activity at 40°C and pH 7.0 and was stable at pH 6.5–10 and temperature around 25°C, retaining over 90 and 80% of initial activity, respectively. RbCCO showed maximum activity at 35°C and pH 9.0 and was stable at pH 6–11 and temperatures of 25–30°C, retaining over 80% of initial activity. The kinetic constants Km of HsCCO for isoeugenol and 4-vinylguaiacol were 1.55 and 1.65 mM and Vmax were 74.09 and 27.91 nmol min–1 mg–1, respectively. The kinetic constants Km of RbCCO for isoeugenol and 4-vinylguaiacol were 2.24 and 0.85 mM and Vmax were 76.48 and 19.96 nmol min–1 mg–1, respectively. The transformed Escherichia coli cells harboring HsCCO converted isoeugenol and 4-vinylguaiacol at molar conversion yields of 80 and 55% and the maximum vanillin concentrations were up to 1.22 and 0.84 g L–1, respectively. Comparably, the molar conversion yields of the transformed E. coli cells harboring RbCCO against isoeugenol 4-vinylguaiacol were 75 and 58%, and the maximum vanillin yields were up to 1.14 and 0.88 g L–1, respectively.

Identification of functionally important residues and structural features in a bacterial lignostilbene dioxygenase

Lignostilbene-α,β-dioxygenase A (LsdA) from the bacterium Sphingomonas paucimobilis TMY1009 is a nonheme iron oxygenase that catalyzes the cleavage of lignostilbene, a compound arising in lignin transformation, to two vanillin molecules. To examine LsdA's substrate specificity, we heterologously produced the dimeric enzyme with the help of chaperones. When tested on several substituted stilbenes, LsdA exhibited the greatest specificity for lignostilbene (kcatapp = 1.00 ± 0.04 × 106 m-1 s-1). These experiments further indicated that the substrate's 4-hydroxy moiety is required for catalysis and that this moiety cannot be replaced with a methoxy group. Phenylazophenol inhibited the LsdA-catalyzed cleavage of lignostilbene in a reversible, mixed fashion (Kic = 6 ± 1 μm, Kiu = 24 ± 4 μm). An X-ray crystal structure of LsdA at 2.3 Å resolution revealed a seven-bladed β-propeller fold with an iron cofactor coordinated by four histidines, in agreement with previous observations on related carotenoid cleavage oxygenases. We noted that residues at the dimer interface are also present in LsdB, another lignostilbene dioxygenase in S. paucimobilis TMY1009, rationalizing LsdA and LsdB homo- and heterodimerization in vivo A structure of an LsdA·phenylazophenol complex identified Phe59, Tyr101, and Lys134 as contacting the 4-hydroxyphenyl moiety of the inhibitor. Phe59 and Tyr101 substitutions with His and Phe, respectively, reduced LsdA activity (kcatapp) ∼15- and 10-fold. The K134M variant did not detectably cleave lignostilbene, indicating that Lys134 plays a key catalytic role. This study expands our mechanistic understanding of LsdA and related stilbene-cleaving dioxygenases.

Mechanistic Insights into a Stibene Cleavage Oxygenase NOV1 from Quantum Mechanical/Molecular Mechanical Calculations.

NOV1, a stilbene cleavage oxygenase, catalyzes the cleavage of the central double bond of stilbenes to two phenolic aldehydes, using a 4‐His Fe(II) center and dioxygen. Herein, we use in‐protein quantum mechanical/molecular mechanical (QM/MM) calculations to elucidate the reaction mechanism of the central double bond cleavage of phytoalexin resveratrol by NOV1. Our results showed that the oxygen molecule prefers to bind to the iron center in a side‐on fashion, as suggested from the experiment. The quintet Fe−O2 complex with the side‐on superoxo antiferromagnetic coupled to the resveratrol radical is identified as the reactive oxygen species. The QM/MM results support the dioxygenase mechanism involving a dioxetane intermediate with a rate‐limiting barrier of 10.0 kcal mol−1. The alternative pathway through an epoxide intermediate is ruled out due to a larger rate‐limiting barrier (26.8 kcal mol−1). These findings provide important insight into the catalytic mechanism of carotenoid cleavage oxygenases and also the dioxygen activation of non‐heme enzymes.

Structure and function of a lignostilbene-\u03b1,\u03b2-dioxygenase orthologue from Pseudomonas brassicacearum

BackgroundStilbene cleaving oxygenases (SCOs), also known as lignostilbene-α,β-dioxygenases (LSDs) mediate the oxidative cleavage of the olefinic double bonds of lignin-derived intermediate phenolic stilbenes, yielding small modified benzaldehyde compounds. SCOs represent one branch of the larger carotenoid cleavage oxygenases family. Here, we describe the structural and functional characterization of an SCO-like enzyme from the soil-born, bio-control agent Pseudomonas brassicacearum.MethodsIn vitro and in vivo assays relying on visual inspection, spectrophotometric quantification, as well as liquid-chormatographic and mass spectrometric characterization were applied for functional evaluation of the enzyme. X-ray crystallographic analyses and in silico modeling were applied for structural investigations.ResultsIn vitro assays demonstrated preferential cleavage of resveratrol, while in vivo analyses detected putative cleavage of the straight chain carotenoid, lycopene. A high-resolution structure containing the seven-bladed β-propeller fold and conserved 4-His-Fe unit at the catalytic site, was obtained. Comparative structural alignments, as well as in silico modelling and docking, highlight potential molecular factors contributing to both the primary in vitro activity against resveratrol, as well as the putative subsidiary activities against carotenoids in vivo, for future validation.ConclusionsThe findings reported here provide validation of the SCO structure, and highlight enigmatic points with respect to the potential effect of the enzyme’s molecular environment on substrate specificities for future investigation.

Preparation and characterization of metal-substituted carotenoid cleavage oxygenases

Carotenoid cleavage oxygenases (CCO) are non-heme iron enzymes that catalyze oxidative cleavage of alkene bonds in carotenoid and stilbenoid substrates. Previously, we showed that the iron cofactor of CAO1, a resveratrol-cleaving member of this family, can be substituted with cobalt to yield a catalytically inert enzyme useful for trapping active site-bound stilbenoid substrates for structural characterization. Metal substitution may provide a general method for identifying the natural substrates for CCOs in addition to facilitating structural and biophysical characterization of CCO-carotenoid complexes under normal aerobic conditions. Here, we demonstrate the general applicability of cobalt substitution in a prototypical carotenoid cleaving CCO, apocarotenoid oxygenase (ACO) from Synechocystis. Among the non-native divalent metals investigated, cobalt was uniquely able to stably occupy the ACO metal binding site and inhibit catalysis. Analysis by X-ray crystallography and X-ray absorption spectroscopy demonstrate that the Co(II) forms of both ACO and CAO1 exhibit a close structural correspondence to the native Fe(II) enzyme forms. Hence, cobalt substitution is an effective strategy for generating catalytically inert but structurally intact forms of CCOs.

Establishment of an Arabidopsis callus system to study the interrelations of biosynthesis, degradation and accumulation of carotenoids.

The net amounts of carotenoids accumulating in plant tissues are determined by the rates of biosynthesis and degradation. While biosynthesis is rate-limited by the activity of PHYTOENE SYNTHASE (PSY), carotenoid losses are caused by catabolic enzymatic and non-enzymatic degradation. We established a system based on non-green Arabidopsis callus which allowed investigating major determinants for high steady-state levels of β-carotene. Wild-type callus development was characterized by strong carotenoid degradation which was only marginally caused by the activity of carotenoid cleavage oxygenases. In contrast, carotenoid degradation occurred mostly non-enzymatically and selectively affected carotenoids in a molecule-dependent manner. Using carotenogenic pathway mutants, we found that linear carotenes such as phytoene, phytofluene and pro-lycopene resisted degradation and accumulated while β-carotene was highly susceptible towards degradation. Moderately increased pathway activity through PSY overexpression was compensated by degradation revealing no net increase in β-carotene. However, higher pathway activities outcompeted carotenoid degradation and efficiently increased steady-state β-carotene amounts to up to 500 μg g-1 dry mass. Furthermore, we identified oxidative β-carotene degradation products which correlated with pathway activities, yielding β-apocarotenals of different chain length and various apocarotene-dialdehydes. The latter included methylglyoxal and glyoxal as putative oxidative end products suggesting a potential recovery of carotenoid-derived carbon for primary metabolic pathways. Moreover, we investigated the site of β-carotene sequestration by co-localization experiments which revealed that β-carotene accumulated as intra-plastid crystals which was confirmed by electron microscopy with carotenoid-accumulating roots. The results are discussed in the context of using the non-green calli carotenoid assay system for approaches targeting high steady-state β-carotene levels prior to their application in crops.