In Vitro Characterization of a Recombinant Blh Protein from an Uncultured Marine Bacterium as a β-Carotene 15,15′-Dioxygenase

Abstract

Codon optimization was used to synthesize the blh gene from the uncultured marine bacterium 66A03 for expression in Escherichia coli. The expressed enzyme cleaved ␤-carotene at its central double bond (15,15) to yield two molecules of all-transretinal. The molecular mass of the native purified enzyme was ϳ64 kDa as a dimer of 32-kDa subunits. The K m , k cat , and k cat /K m values for ␤-carotene as substrate were 37 M, 3.6 min ؊1 , and 97 mM ؊1 min ؊1 , respectively. The enzyme exhibited the highest activity for ␤-carotene, followed by ␤-cryptoxanthin, ␤-apo-4-carotenal, ␣-carotene, and ␥-carotene in decreasing order, but not for ␤-apo-8-carotenal, ␤-apo-12-carotenal, lutein, zeaxanthin, or lycopene, suggesting that the presence of one unsubstituted ␤-ionone ring in a substrate with a molecular weight greater than C 35 seems to be essential for enzyme activity. The oxygen atom of retinal originated not from water but from molecular oxygen, suggesting that the enzyme was a ␤-carotene 15,15-dioxygenase. Although the Blh protein and ␤-carotene 15,15-monooxygenases catalyzed the same biochemical reaction, the Blh protein was unrelated to the mammalian ␤-carotene 15,15-monooxygenases as assessed by their different properties, including DNA and amino acid sequences, molecular weight, form of association, reaction mechanism, kinetic properties, and substrate specificity. This is the first report of in vitro characterization of a bacterial ␤-carotene-cleaving enzyme.Vitamin A (retinol) is a fat-soluble vitamin and important for human health. In vivo, the cleavage of ␤-carotene to retinal is an important step of vitamin A synthesis. The cleavage can proceed via two different biochemical pathways (1, 2). The major pathway is a central cleavage catalyzed by mammalian ␤-carotene 15,15Ј-monooxygenases (EC 1.14.99.36). ␤-Carotene is cleaved by the enzyme symmetrically into two molecules of alltrans-retinal, and retinal is then converted to vitamin A in vivo (3-5). The second pathway is an eccentric cleavage that occurs at double bonds other than the central 15,15Ј-double bond of ␤-carotene to produce ␤-apo-carotenals with different chain lengths, which are catalyzed by carotenoid oxygenases from mammals, plants, and cyanobacteria (6). These ␤-apo-carotenals are degraded to one molecule of retinal, which is subsequently converted to vitamin A in vivo (2).␤-Carotene 15,15Ј-monooxygenase was first isolated as a cytosolic enzyme by identifying the product of ␤-carotene cleavage as retinal (7). The characterization of the enzyme and the reaction pathway from ␤-carotene to retinal were also investigated (4, 8). The enzyme activity has been found in mammalian intestinal mucosa, jejunum enterocytes, liver, lung, kidney, and brain (5, 9, 10). Molecular cloning, expression, and characterization of ␤-carotene 15,15Ј-monooxygenase have been reported from various species, including chickens (11), fruit flies (12), humans (13), mice (14), and zebra fishes (15).Other proteins thought to convert ␤-carotene to retinal include bacterioopsin-r...

The Human Enzyme That Converts Dietary Provitamin A Carotenoids to Vitamin A Is a Dioxygenase

“…For the BCO1-␤-carotene reaction in 16 O 2 -H 2 18 O medium, the retinal product obtained (approximately 60 pmol) after a 15-min reaction was only about 3-10% 18 O-retinal (Fig. 1C).…”

“…Quantification of Retinal Oxygen Isotopologues-The fragmentation patterns of 18 O-retinal and 16 O-retinal are virtually the same (see Fig. 3).…”

“…These daughter ions were selected because they were the dominant fragment ions. Also, the last three daughter ions listed for 18 Oretinal and 16 O-retinal differ by 2 atomic mass units, indicating that these fragments bear the oxygen atom.…”

“…Synthesis of 18 O-retinal-All-trans-retinal (20 nmol), H 2 18 O (200 equivalents), 2 ml of acetonitrile, and 60 mg of Dowex 50W-X4 (hydrogen form) were stirred at room temperature in a closed vial protected from light for 1.5 h. This is based on the method of Kawanishi et al (15). The solids were removed by decantation, and the retinoids were then extracted with 3 ϫ 2-ml hexanes.…”

“…The MS instrumental parameters included: sheath gas temperature, 400°C; flow rate, 12 liters/min; drying gas temperature, 150°C; flow rate, 15 liters/min; 3-Hz MS/MS acquisition; 10-Hz MS reference scans; 30 psig nebulizer; Vcap, 2000 V; nozzle voltage, 2000 V; fragmentor, 350 V; ion funnel settings for small molecules. MS/MS transitions were acquired by collision-induced dissociation of all-trans-retinal standard (m/z ϭ 285.218) and 18 O-retinal (m/z ϭ 287.226) and found to optimize at a collision energy of 7.5 eV. Source and collision-induced dissociation gas was high purity (Ͼ98%) nitrogen.…”