title: Substrate Specificity of Purified Recombinant Chicken β-Carotene 9',10'-Oxygenase (BCO2).
abstract: Provitamin A carotenoids are oxidatively cleaved by β-carotene 15,15'-dioxygenase (BCO1) at the central 15-15' double bond to form retinal (vitamin A aldehyde). Another carotenoid oxygenase, β-carotene 9',10'-oxygenase (BCO2) catalyzes the oxidative cleavage of carotenoids at the 9'-10' bond to yield an ionone and an apo-10'-carotenoid. Previously published substrate specificity studies of BCO2 were conducted using crude lysates from bacteria or insect cells expressing recombinant BCO2. Our attempts to obtain active recombinant human BCO2 expressed in Escherichia coli were unsuccessful. We have expressed recombinant chicken BCO2 in the strain E. coli BL21-Gold (DE3) and purified the enzyme by cobalt ion affinity chromatography. Like BCO1, purified recombinant chicken BCO2 catalyzes the oxidative cleavage of the provitamin A carotenoids β-carotene, α-carotene, and β-cryptoxanthin. Its catalytic activity with β-carotene as substrate is at least 10-fold lower than that of BCO1. In further contrast to BCO1, purified recombinant chicken BCO2 also catalyzes the oxidative cleavage of 9-cis-β-carotene and the non-provitamin A carotenoids zeaxanthin and lutein, and is inactive with all-trans-lycopene and β-apocarotenoids. Apo-10'-carotenoids were detected as enzymatic products by HPLC, and the identities were confirmed by LC-MS. Small amounts of 3-hydroxy-β-apo-8'-carotenal were also consistently detected in BCO2-β-cryptoxanthin reaction mixtures. With the exception of this activity with β-cryptoxanthin, BCO2 cleaves specifically at the 9'-10' bond to produce apo-10'-carotenoids. BCO2 has been shown to function in preventing the excessive accumulation of carotenoids, and its broad substrate specificity is consistent with this.
authors.affiliation: From the Department of Human Nutrition, Ohio State Biochemistry Program.
authors.forename: Carlo
authors.initials: C
authors.lastname: Dela Seña
authors.affiliation: From the Department of Human Nutrition.
authors.forename: Jian
authors.initials: J
authors.lastname: Sun
authors.affiliation: From the Department of Human Nutrition.
authors.forename: Sureshbabu
authors.initials: S
authors.lastname: Narayanasamy
authors.affiliation: Department of Food Science and Technology, and.
authors.forename: Kenneth M
authors.initials: KM
authors.lastname: Riedl
authors.affiliation: From the Department of Human Nutrition.
authors.forename: Yan
authors.initials: Y
authors.lastname: Yuan
authors.affiliation: Ohio State Biochemistry Program, College of Pharmacy, The Ohio State University, Columbus, Ohio 43210.
authors.forename: Robert W
authors.initials: RW
authors.lastname: Curley
authors.affiliation: Department of Food Science and Technology, and.
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Schwartz
authors.affiliation: From the Department of Human Nutrition, Ohio State Biochemistry Program, harrison.304@osu.edu.
authors.forename: Earl H
authors.initials: EH
authors.lastname: Harrison
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002338:Carotenoids / Q000737:chemistry / Q000378:metabolism; D002645:Chickens / Q000235:genetics / Q000378:metabolism*; D065366:Cryptoxanthins / Q000737:chemistry / Q000378:metabolism; D049308:Dioxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D019207:beta Carotene / Q000737:chemistry / Q000378:metabolism*
chemical_list: D065366:Cryptoxanthins; D011994:Recombinant Proteins; C110652:beta-apo-8'-carotenal; D019207:beta Carotene; D002338:Carotenoids; C041635:alpha-carotene; D049308:Dioxygenases; C506534:BCO2 protein, human
keywords: carotenoid; dioxygenase; enzyme; enzyme purification; lipid metabolism; recombinant protein expression; retinoid