Abdulkerim Eroglu, Earl H. Harrison

β-Carotene is the major dietary source of provitamin A. Central cleavage of β-carotene catalyzed by β-carotene oxygenase 1 yields two molecules of retinaldehyde. Subsequent oxidation produces all-trans-retinoic acid (ATRA), which functions as a ligand for a family of nuclear transcription factors, the retinoic acid receptors (RARs). Eccentric cleavage of β-carotene at non-central double bonds is catalyzed by other enzymes and can also occur non-enzymatically. The products of these reactions are β-apocarotenals and β-apocarotenones, whose biological functions in mammals are unknown. We used reporter gene assays to show that none of the β-apocarotenoids significantly activated RARs. Importantly, however, β-apo-14'-carotenal, β-apo-14'-carotenoic acid, and β-apo-13-carotenone antagonized ATRA-induced transactivation of RARs. Competitive radioligand binding assays demonstrated that these putative RAR antagonists compete directly with retinoic acid for high affinity binding to purified receptors. Molecular modeling studies confirmed that β-apo-13-carotenone can interact directly with the ligand binding site of the retinoid receptors. β-Apo-13-carotenone and the β-apo-14'-carotenoids inhibited ATRA-induced expression of retinoid responsive genes in Hep G2 cells. Finally, we developed an LC/MS method and found 3-5 nm β-apo-13-carotenone was present in human plasma. These findings suggest that β-apocarotenoids function as naturally occurring retinoid antagonists. The antagonism of retinoid signaling by these metabolites may have implications for the activities of dietary β-carotene as a provitamin A and as a modulator of risk for cardiovascular disease and cancer.

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.

Apocarotenoids are cleavage products of C40 isoprenoid pigments, named carotenoids, synthesized exclusively by plants and microorganisms. The colors of flowers and fruits and the photosynthetic process are examples of the biological properties conferred by carotenoids to these organisms. Mammals do not synthesize carotenoids but obtain them from foods of plant origin. Apocarotenoids are generated upon enzymatic and nonenzymatic cleavage of the parent compounds both in plants and in the tissues of mammals that have ingested carotenoid-containing foods. The best-characterized apocarotenoids are retinoids (vitamin A and its derivatives), generated upon central oxidative cleavage of provitamin A carotenoids, mainly β-carotene. In addition to the well-known biological actions of vitamin A, it is becoming apparent that nonretinoid apocarotenoids also have the potential to regulate a broad spectrum of critical cellular functions, thus influencing mammalian health. This review discusses the current knowledge about the generation and biological activities of nonretinoid apocarotenoids in mammals.