title: Substrate specificity of purified recombinant human β-carotene 15,15'-oxygenase (BCO1).
abstract: Humans cannot synthesize vitamin A and thus must obtain it from their diet. β-Carotene 15,15'-oxygenase (BCO1) catalyzes the oxidative cleavage of provitamin A carotenoids at the central 15-15' double bond to yield retinal (vitamin A). In this work, we quantitatively describe the substrate specificity of purified recombinant human BCO1 in terms of catalytic efficiency values (kcat/Km). The full-length open reading frame of human BCO1 was cloned into the pET-28b expression vector with a C-terminal polyhistidine tag, and the protein was expressed in the Escherichia coli strain BL21-Gold(DE3). The enzyme was purified using cobalt ion affinity chromatography. The purified enzyme preparation catalyzed the oxidative cleavage of β-carotene with a Vmax = 197.2 nmol retinal/mg BCO1 × h, Km = 17.2 μM and catalytic efficiency kcat/Km = 6098 M(-1) min(-1). The enzyme also catalyzed the oxidative cleavage of α-carotene, β-cryptoxanthin, and β-apo-8'-carotenal to yield retinal. The catalytic efficiency values of these substrates are lower than that of β-carotene. Surprisingly, BCO1 catalyzed the oxidative cleavage of lycopene to yield acycloretinal with a catalytic efficiency similar to that of β-carotene. The shorter β-apocarotenals (β-apo-10'-carotenal, β-apo-12'-carotenal, β-apo-14'-carotenal) do not show Michaelis-Menten behavior under the conditions tested. We did not detect any activity with lutein, zeaxanthin, and 9-cis-β-carotene. Our results show that BCO1 favors full-length provitamin A carotenoids as substrates, with the notable exception of lycopene. Lycopene has previously been reported to be unreactive with BCO1, and our findings warrant a fresh look at acycloretinal and its alcohol and acid forms as metabolites of lycopene in future studies.
authors.affiliation: From the Department of Human Nutrition.
authors.forename: Carlo
authors.initials: C
authors.lastname: dela Seña
authors.forename: Sureshbabu
authors.initials: S
authors.lastname: Narayanasamy
authors.forename: Kenneth M
authors.initials: KM
authors.lastname: Riedl
authors.forename: Robert W
authors.initials: RW
authors.lastname: Curley
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Schwartz
authors.forename: Earl H
authors.initials: EH
authors.lastname: Harrison
mesh_terms: D002338:Carotenoids / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D006801:Humans; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D050559:beta-Carotene 15,15'-Monooxygenase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D002338:Carotenoids; C493358:BCO1 protein, human; D050559:beta-Carotene 15,15'-Monooxygenase
keywords: Acycloretinal; Apocarotenal; Carotenoid; Enzymes; Lipid Metabolism; Lycopene; Metabolism; Retinal; Retinoid; Vitamin A