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Structural basis for carotenoid cleavage by an archaeal carotenoid dioxygenase.

ABSTRACT: Apocarotenoids are important signaling molecules generated from carotenoids through the action of carotenoid cleavage dioxygenases (CCDs). These enzymes have a remarkable ability to cleave carotenoids at specific alkene bonds while leaving chemically similar sites within the polyene intact. Although several bacterial and eukaryotic CCDs have been characterized, the long-standing goal of experimentally visualizing a CCD-carotenoid complex at high resolution to explain this exquisite regioselectivity remains unfulfilled. CCD genes are also present in some archaeal genomes, but the encoded enzymes remain uninvestigated. Here, we address this knowledge gap through analysis of a metazoan-like archaeal CCD from Candidatus Nitrosotalea devanaterra (NdCCD). NdCCD was active toward ?-apocarotenoids but did not cleave bicyclic carotenoids. It exhibited an unusual regiospecificity, cleaving apocarotenoids solely at the C14'-C13' alkene bond to produce ?-apo-14'-carotenals. The structure of NdCCD revealed a tapered active site cavity markedly different from the broad active site observed for the retinal-forming Synechocystis apocarotenoid oxygenase (SynACO) but similar to the vertebrate retinoid isomerase RPE65. The structure of NdCCD in complex with its apocarotenoid product demonstrated that the site of cleavage is defined by interactions along the substrate binding cleft as well as selective stabilization of reaction intermediates at the scissile alkene. These data on the molecular basis of CCD catalysis shed light on the origins of the varied catalytic activities found in metazoan CCDs, opening the possibility of modifying their activity through rational chemical or genetic approaches.

SUBMITTER: Daruwalla A

PROVIDER: S-EPMC7443882 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Structural basis for carotenoid cleavage by an archaeal carotenoid dioxygenase.

Daruwalla Anahita A Zhang Jianye J Lee Ho Jun HJ Khadka Nimesh N Farquhar Erik R ER Shi Wuxian W von Lintig Johannes J Kiser Philip D PD

Proceedings of the National Academy of Sciences of the United States of America 20200803 33

Apocarotenoids are important signaling molecules generated from carotenoids through the action of carotenoid cleavage dioxygenases (CCDs). These enzymes have a remarkable ability to cleave carotenoids at specific alkene bonds while leaving chemically similar sites within the polyene intact. Although several bacterial and eukaryotic CCDs have been characterized, the long-standing goal of experimentally visualizing a CCD-carotenoid complex at high resolution to explain this exquisite regioselectiv ...[more]

PMID: 32747548

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Project description:The dietary needs of humans for provitamin A carotenoids arise from their inability to synthesize vitamin A de novo. To improve the status of this essential micronutrient, special attention has been given to biofortification of staple foods, such as wheat grains, which are consumed in large quantities but contain low levels of provitamin A carotenoids. However, there remains an unclear contribution of metabolic genes and homoeologs to the turnover of carotenoids in wheat grains. To better understand carotenoid catabolism in tetraploid wheat, Targeting Induced Local Lesions in Genomes (TILLING) mutants of CCD4, encoding a Carotenoid Cleavage Dioxygenase (CCD) that cleaves carotenoids into smaller apocarotenoid molecules, were isolated and characterized. Our analysis showed that ccd4 mutations co-segregated with Poltergeist-like (pll) mutations in the TILLING mutants of A and B subgenomes, hence the ccd-A4 pll-A, ccd-B4 pll-B, and ccd-A4 ccd-B4 pll-A pll-B mutants were analyzed in this study. Carotenoid profiles are comparable in mature grains of the mutant and control plants, indicating that CCD4 homoeologs do not have a major impact on carotenoid accumulation in grains. However, the neoxanthin content was increased in leaves of ccd-A4 ccd-B4 pll-A pll-B relative to the control. In addition, four unidentified carotenoids showed a unique presence in leaves of ccd-A4 ccd-B4 pll-A pll-B plants. These results suggested that CCD4 homoeologs may contribute to the turnover of neoxanthin and the unidentified carotenoids in leaves. Interestingly, abnormal spike, grain, and seminal root phenotypes were also observed for ccd-A4 pll-A, ccd-B4 pll-B, and ccd-A4 ccd-B4 pll-A pll-B plants, suggesting that CCD4 and/or PLL homoeologs could function toward these traits. Overall, this study not only reveals the role of CCD4 in cleavage of carotenoids in leaves and grains, but also uncovers several critical growth traits that are controlled by CCD4, PLL, or the CCD4-PLL interaction.

Project description:BackgroundIn plants, carotenoids serve as the precursors to C13-norisoprenoids, a group of apocarotenoid compounds with diverse biological functions. Enzymatic cleavage of carotenoids catalysed by members of the carotenoid cleavage dioxygenase (CCD) family has been shown to produce a number of industrially important volatile flavour and aroma apocarotenoids including β-ionone, geranylacetone, pseudoionone, α-ionone and 3-hydroxy-β-ionone in a range of plant species. Apocarotenoids contribute to the floral and fruity attributes of many wine cultivars and are thereby, at least partly, responsible for the "varietal character". Despite their importance in grapes and wine; carotenoid cleavage activity has only been described for VvCCD1 and the mechanism(s) and regulation of carotenoid catabolism remains largely unknown.ResultsThree grapevine-derived CCD-encoding genes have been isolated and shown to be functional with unique substrate cleavage capacities. Our results demonstrate that the VvCCD4a and VvCCD4b catalyse the cleavage of both linear and cyclic carotenoid substrates. The expression of VvCCD1, VvCCD4a and VvCCD4b was detected in leaf, flower and throughout berry development. VvCCD1 expression was constitutive, whereas VvCCD4a expression was predominant in leaves and VvCCD4b in berries. A transgenic population with a 12-fold range of VvCCD1 expression exhibited a lack of correlation between VvCCD1 expression and carotenoid substrates and/or apocarotenoid products in leaves, providing proof that the in planta function(s) of VvCCD1 in photosynthetically active tissue is distinct from the in vitro activities demonstrated. The isolation and functional characterisation of VvCCD4a and VvCCD4b identify two additional CCDs that are functional in grapevine.ConclusionsTaken together, our results indicate that the three CCDs are under various levels of control that include gene expression (spatial and temporal), substrate specificity and compartmentalisation that act individually and/or co-ordinately to maintain carotenoid and volatile apocarotenoid levels in plants. Altering the expression of VvCCD1 in a transgenic grapevine population illustrated the divergence between the in vitro enzyme activity and the in planta activity of this enzyme, thereby contributing to the efforts to understand how enzymatic degradation of carotenoids involved in photosynthesis occurs. The identification and functional characterisation of VvCCD4a and VvCCD4b suggest that these enzymes are primarily responsible for catalysing the cleavage of plastidial carotenoids.

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Structural basis for carotenoid cleavage by an archaeal carotenoid dioxygenase.

Publications

Structural basis for carotenoid cleavage by an archaeal carotenoid dioxygenase.

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