title: Analysis of carotenoid isomerase activity in a prototypical carotenoid cleavage enzyme, apocarotenoid oxygenase (ACO).
abstract: Carotenoid cleavage enzymes (CCEs) constitute a group of evolutionarily related proteins that metabolize a variety of carotenoid and non-carotenoid substrates. Typically, these enzymes utilize a non-heme iron center to oxidatively cleave a carbon-carbon double bond of a carotenoid substrate. Some members also isomerize specific double bonds in their substrates to yield cis-apocarotenoid products. The apocarotenoid oxygenase from Synechocystis has been hypothesized to represent one such member of this latter category of CCEs. Here, we developed a novel expression and purification protocol that enabled production of soluble, native ACO in quantities sufficient for high resolution structural and spectroscopic investigation of its catalytic mechanism. High performance liquid chromatography and Raman spectroscopy revealed that ACO exclusively formed all-trans products. We also found that linear polyoxyethylene detergents previously used for ACO crystallization strongly inhibited the apocarotenoid oxygenase activity of the enzyme. We crystallized the native enzyme in the absence of apocarotenoid substrate and found electron density in the active site that was similar in appearance to the density previously attributed to a di-cis-apocarotenoid intermediate. Our results clearly demonstrated that ACO is in fact a non-isomerizing member of the CCE family. These results indicate that careful selection of detergent is critical for the success of structural studies aimed at elucidating structures of CCE-carotenoid/retinoid complexes.
authors.affiliation: From the Departments of Pharmacology and.
authors.forename: Xuewu
authors.initials: X
authors.lastname: Sui
authors.forename: Philip D
authors.initials: PD
authors.lastname: Kiser
authors.forename: Tao
authors.initials: T
authors.lastname: Che
authors.forename: Paul R
authors.initials: PR
authors.lastname: Carey
authors.forename: Marcin
authors.initials: M
authors.lastname: Golczak
authors.forename: Wuxian
authors.initials: W
authors.lastname: Shi
authors.forename: Johannes
authors.initials: J
authors.lastname: von Lintig
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Palczewski
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis / Q000187:drug effects; D002338:Carotenoids / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D018360:Crystallography, X-Ray; D007535:Isomerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000737:chemistry / Q000494:pharmacology; D012172:Retinaldehyde / Q000737:chemistry / Q000378:metabolism; D013059:Spectrum Analysis, Raman; D046940:Synechococcus / Q000201:enzymology / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D002338:Carotenoids; D011092:Polyethylene Glycols; D010105:Oxygenases; C524249:apocarotenal oxygenase, Synechocystis; D007535:Isomerases; D012172:Retinaldehyde
keywords: Carotene; Carotenoid; Enzyme Catalysis; Enzyme Inactivation; Enzyme Mechanisms; Enzyme Structure; Vitamin A