Utilization of Dioxygen by Carotenoid Cleavage Oxygenases.
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 (18)O-labeled water and (18)O2 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.
From the Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4956 and.
Xuewu
X
Sui
From the Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4956 and.
Marcin
M
Golczak
From the Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4956 and.
Jianye
J
Zhang
From the Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4956 and.
Katie A
KA
Kleinberg
From the Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4956 and.
Johannes
J
von Lintig
From the Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4956 and kxp65@case.edu.
Krzysztof
K
Palczewski
From the Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4956 and the Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, Ohio 44106 pdk7@case.edu.
Philip D
PD
Kiser
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002338:Carotenoids / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D049308:Dioxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019746:Intramolecular Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010100:Oxygen / Q000737:chemistry* / Q000378:metabolism; D000077185:Resveratrol; D013267:Stilbenes / Q000737:chemistry* / Q000378:metabolism; D046939:Synechocystis / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D013267:Stilbenes; D002338:Carotenoids; D049308:Dioxygenases; D019746:Intramolecular Oxidoreductases; D000077185:Resveratrol; D010100:Oxygen
carotenoid; enzyme; enzyme catalysis; enzyme mechanism; enzyme structure; retinal metabolism; retinoid; retinol