Journal of Biological Chemistry
Volume 290, Issue 51, 18 December 2015, Pages 30212-30223
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Enzymology
Utilization of Dioxygen by Carotenoid Cleavage Oxygenases*

https://doi.org/10.1074/jbc.M115.696799Get rights and content
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Carotenoid cleavage oxygenases (CCOs) are non-heme, Fe(II)-dependent enzymes that participate in biologically important metabolic pathways involving carotenoids and apocarotenoids, including retinoids, stilbenes, and related compounds. CCOs typically catalyze the cleavage of non-aromatic double bonds by dioxygen (O2) to form aldehyde or ketone products. Expressed only in vertebrates, the RPE65 sub-group of CCOs catalyzes a non-canonical reaction consisting of concerted ester cleavage and trans-cis isomerization of all-trans-retinyl esters. It remains unclear whether the former group of CCOs functions as mono- or di-oxygenases. Additionally, a potential role for O2 in catalysis by the RPE65 group of CCOs has not been evaluated to date. Here, we investigated the pattern of oxygen incorporation into apocarotenoid products of Synechocystis apocarotenoid oxygenase. Reactions performed in the presence of 18O-labeled water and 18O2 revealed an unambiguous dioxygenase pattern of O2 incorporation into the reaction products. Substitution of Ala for Thr at position 136 of apocarotenoid oxygenase, a site predicted to govern the mono- versus dioxygenase tendency of CCOs, greatly reduced enzymatic activity without altering the dioxygenase labeling pattern. Reevaluation of the oxygen-labeling pattern of the resveratrol-cleaving CCO, NOV2, previously reported to be a monooxygenase, using a purified enzyme sample revealed that it too is a dioxygenase. We also demonstrated that bovine RPE65 is not dependent on O2 for its cleavage/isomerase activity. In conjunction with prior research, the results of this study resolve key issues regarding the utilization of O2 by CCOs and indicate that dioxygenase activity is a feature common among double bond-cleaving CCOs.

carotenoid
enzyme
enzyme catalysis
enzyme mechanism
enzyme structure
retinal metabolism
retinoid
retinol

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The atomic coordinates and structure factors (code 5E47) have been deposited in the Protein Data Bank (http://wwpdb.org/).

*

This work was supported by Department of Veterans Affairs Career Development Award IK2BX002683 (to P. D. K.) and National Institutes of Health Grants EY009339 (to K. P.), EY023948 (to M. G.), and EY020551 (to K. P. and J. v. L.). A portion of this work is based upon research conducted at the Advanced Photon National Institutes of Health Grant GM103403 from the NCRR. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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