Publications

• In vitro characterization of a recombinant Blh protein from an uncultured marine bacterium as a beta-carotene 15,15'-dioxygenase.

  Kim Y.S., Kim N.H., Yeom S.J., Kim S.W., Oh D.K.

  Codon optimization was used to synthesize the blh gene from the uncultured marine bacterium 66A03 for expression in Escherichia coli. The expressed enzyme cleaved beta-carotene at its central double bond (15,15') to yield two molecules of all-trans-retinal. The molecular mass of the native purifie ... >> More

  Codon optimization was used to synthesize the blh gene from the uncultured marine bacterium 66A03 for expression in Escherichia coli. The expressed enzyme cleaved beta-carotene at its central double bond (15,15') to yield two molecules of all-trans-retinal. The molecular mass of the native purified enzyme was approximately 64 kDa as a dimer of 32-kDa subunits. The K(m), k(cat), and k(cat)/K(m) values for beta-carotene as substrate were 37 mum, 3.6 min(-1), and 97 mm(-1) min(-1), respectively. The enzyme exhibited the highest activity for beta-carotene, followed by beta-cryptoxanthin, beta-apo-4'-carotenal, alpha-carotene, and gamma-carotene in decreasing order, but not for beta-apo-8'-carotenal, beta-apo-12'-carotenal, lutein, zeaxanthin, or lycopene, suggesting that the presence of one unsubstituted beta-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 beta-carotene 15,15'-dioxygenase. Although the Blh protein and beta-carotene 15,15'-monooxygenases catalyzed the same biochemical reaction, the Blh protein was unrelated to the mammalian beta-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 beta-carotene-cleaving enzyme. << Less

  J. Biol. Chem. 284:15781-15793(2009) [PubMed] [EuropePMC]

• Retinal production from beta-carotene by beta-carotene 15,15'-dioxygenase from an unculturable marine bacterium.

  Kim Y.S., Park C.S., Oh D.K.

  The conversion of beta-carotene to retinal by a recombinant beta-carotene 15,15'-dioxygenase (Blh protein) from an unculturable marine bacterium was optimized in aqueous solution. Toluene was optimal solvent for the dissolution of beta-carotene and the optimal solution for the conversion reaction ... >> More

  The conversion of beta-carotene to retinal by a recombinant beta-carotene 15,15'-dioxygenase (Blh protein) from an unculturable marine bacterium was optimized in aqueous solution. Toluene was optimal solvent for the dissolution of beta-carotene and the optimal solution for the conversion reaction contained 2.4% (w/v) Tween 20, 0.15 U enzyme/ml, and 350 mg beta-carotene/l. Under these conditions, the enzyme produced 181 mg retinal/l after 20 h. This is the highest reported value for the retinal concentration from beta-carotene. << Less

  Biotechnol. Lett. 32:957-961(2010) [PubMed] [EuropePMC]

• Substrate specificity of a recombinant chicken beta-carotene 15,15'-monooxygenase that converts beta-carotene into retinal.

  Kim Y.S., Oh D.K.

  The recombinant beta-carotene 15,15'-monooxygenase from chicken liver was purified as a single 60 kDa band by His-Trap HP and Resource Q chromatography. It had a molecular mass of 240 kDa by gel filtration indicating the native form to be tetramer. The enzyme converted beta-carotene under maximal ... >> More

  The recombinant beta-carotene 15,15'-monooxygenase from chicken liver was purified as a single 60 kDa band by His-Trap HP and Resource Q chromatography. It had a molecular mass of 240 kDa by gel filtration indicating the native form to be tetramer. The enzyme converted beta-carotene under maximal conditions (pH 8.0 and 37 degrees C) with a k (cat) of 1.65 min(-1) and a K (m) of 26 muM and its conversion yield of beta-carotene to retinal was 120% (mol mol(-1)). The enzyme displayed catalytic efficiency and conversion yield for beta-carotene, beta-cryptoxanthin, beta-apo-8'-carotenal, beta-apo-4'-carotenal, alpha-carotene and gamma-carotene in decreasing order but not for zeaxanthin, lutein, beta-apo-12'-carotenal and lycopene, suggesting that the presence of one unsubstituted beta-ionone ring in a substrate with a molecular weight greater than C(30) seems to be essential for enzyme activity. << Less

  Biotechnol Lett 31:403-408(2009) [PubMed] [EuropePMC]

• Substrate specificity of purified recombinant human beta-carotene 15,15'-oxygenase (BCO1).

  dela Sena C., Narayanasamy S., Riedl K.M., Curley R.W. Jr., Schwartz S.J., Harrison E.H.

  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 specifi ... >> More

  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. << Less

  J Biol Chem 288:37094-37103(2013) [PubMed] [EuropePMC]

• The human enzyme that converts dietary provitamin A carotenoids to vitamin A is a dioxygenase.

  dela Sena C., Riedl K.M., Narayanasamy S., Curley R.W. Jr., Schwartz S.J., Harrison E.H.

  β-Carotene 15-15'-oxygenase (BCO1) catalyzes the oxidative cleavage of dietary provitamin A carotenoids to retinal (vitamin A aldehyde). Aldehydes readily exchange their carbonyl oxygen with water, making oxygen labeling experiments challenging. BCO1 has been thought to be a monooxygenase, incorpo ... >> More

  β-Carotene 15-15'-oxygenase (BCO1) catalyzes the oxidative cleavage of dietary provitamin A carotenoids to retinal (vitamin A aldehyde). Aldehydes readily exchange their carbonyl oxygen with water, making oxygen labeling experiments challenging. BCO1 has been thought to be a monooxygenase, incorporating oxygen from O2 and H2O into its cleavage products. This was based on a study that used conditions that favored oxygen exchange with water. We incubated purified recombinant human BCO1 and β-carotene in either (16)O2-H2(18)O or (18)O2-H2(16)O medium for 15 min at 37 °C, and the relative amounts of (18)O-retinal and (16)O-retinal were measured by liquid chromatography-tandem mass spectrometry. At least 79% of the retinal produced by the reaction has the same oxygen isotope as the O2 gas used. Together with the data from (18)O-retinal-H2(16)O and (16)O-retinal-H2(18)O incubations to account for nonenzymatic oxygen exchange, our results show that BCO1 incorporates only oxygen from O2 into retinal. Thus, BCO1 is a dioxygenase. << Less

  J. Biol. Chem. 289:13661-13666(2014) [PubMed] [EuropePMC]