Identification, Expression, and Substrate Specificity of a Mammalian β-Carotene 15,15′-Dioxygenase

Abstract

We have identified from mouse the first mammalian ␤-carotene 15,15-dioxygenase (␤-CD), a crucial enzyme in development and metabolism that governs the de novo entry of vitamin A from plant-derived precursors. ␤-CD is related to the retinal pigment epithelium-expressed protein RPE65 and belongs to a diverse family that includes the plant 9-cis-epoxycarotenoid dioxygenase and bacterial lignostilbene dioxygenases. ␤-CD expression in Escherichia coli cells engineered to produce ␤-carotene led to the accumulation of all-trans-retinal at the expense of ␤-carotene, confirming that ␤-CD catalyzed the central cleavage of this vitamin A precursor. Purified recombinant ␤-CD protein cleaves ␤-carotene in vitro with a V max of 36 pmol of retinal/mg of enzyme/ min and a K m of 6 M. Non-provitamin A carotenoids were also cleaved, although with much lower activity. By Northern analysis, a 2.4-kilobase (kb) message was observed in liver, kidney, small intestine, and testis, tissues important in retinoid/carotenoid metabolism. This message encoded a 63-kDa cytosolic protein expressed in these tissues. A shorter transcript of 1.8 kb was found in testis and skin. Developmentally, the 2.4-kb mRNA was abundant at embryonic day 7, with lower expression at embryonic days 11, 13, and 15, suggesting a critical role for this enzyme in gastrulation. Identification of ␤-CD in an accessible model organism will create new opportunities to study vitamin A metabolism.In vertebrates, vitamin A in its various oxidative and isomeric forms is essential for embryonic development (1), pattern formation (2, 3), and vision (4). Retinoic acid, through its interaction with the nuclear retinoic acid receptor and retinoid X receptor, profoundly affects cell differentiation and development. Because animals are unable to synthesize vitamin A de novo from endogenous isoprenoid precursors, they must instead derive it from cleavage of ␤-carotene and certain other carotenoids with an unsubstituted ␤-ring (e.g. ␥-and ␣-carotenes, ␤-zeacarotene, and ␤-cryptoxanthin). It is generally accepted that central cleavage of ␤-carotene by a putative dioxygenase gives rise to two molecules of all-trans-retinal, whereas eccentric cleavage with subsequent processing leading to a single molecule of retinoic acid from an apocarotenal is quantitatively far less important (5). ␤-Carotene cleavage activity is reported highest in the intestinal mucosa, but is found at high activity levels in liver, kidney, lung, and fat tissues, among other sites. However, an inability to purify the protein catalyzing this reaction has hindered thorough investigation of this crucial first step in vitamin A metabolism.Because of a loose similarity between the mammalian protein RPE65 and the neoxanthin cleavage enzymes of plants, our laboratories have considered the hypothesis that the putative ␤-CD 1 would belong to an emerging family of carotenoidcleaving dioxygenases known mainly from examples in plants (6), but with members also in bacteria and Metazoa. The first described representative was a ba...

Drosophila NinaB and NinaD Act Outside of Retina to Produce Rhodopsin Chromophore

“…The requirement of ␤-carotene as the C 40 carotenoid acted on by the NinaB BCO is suggested by the observation that bacterially expressed NinaB BCO does not process zeaxanthin or other hydroxylated C 40 carotenoids to the C 20 3-OH retinal (26). Similar specificity was observed for mammalian BCO homologs (12,13). If Drosophila in vivo conditions of NinaB BCO activity are the same, the successful use of zeaxanthin or lutein (16) as a dietary source for the 3-OH retinal requires that these hydroxylated C 40 carotenoids are first converted to ␤-carotene.…”

“…The availability of vitamin A for metabolic processes is governed by multiple factors, including dietary absorption, transport, metabolism, and storage (11). A human BCO is expressed in the retinal pigment epithelium and also in the kidney, intestine, liver, brain, stomach, and testis (12,13), suggesting that the processing of dietary carotenoids occurs in a variety of vertebrate tissues. Additional studies show that centric cleavage of ␤-carotene plays a major role in the processing of carotenoids (14,15).…”

Vertebrate and invertebrate carotenoid-binding proteins

“…Although beta-carotene cleavage activity was demonstrated in cell-free extracts derived from mammalian small intestine in the 1960s, purification of the enzyme proved surprisingly challenging [47][48][49][50]. It was not until the advent of molecular biological approaches and mass spectral sequencing that the beta-carotene cleavage enzyme was cloned first from Drosophila and chicken and later from humans [54][55][56][57][58]. It is a cytosolic enzyme primarily localized in the duodenal mucosa although it has been found in liver and in the eye.…”

“…It has been demonstrated that this enzyme can act on other carotenoids also; however, provitamin A activity might vary depending on the substrate. Cloning of mammalian proteins similar to the 15, 15′ β-carotene oxygenase has revealed the existence of RPE65 [54,56], a protein critical to the cis-trans isomerization of retinoids in the visual cycle, as well as an enzyme that catalyzes the asymmetric cleavage of beta-carotene at the 9′-10′ position [47][48][49]57].…”

Mutation of key residues of RPE65 abolishes its enzymatic role as isomerohydrolase in the visual cycle

“…Alternatively, because it belongs to a family of carotenoid oxygenases, RPE65 may play a direct catalytic role in the visual cycle. This family, including bacterial (22), plant (23), invertebrate (24), and vertebrate (25,26) carotenoid oxygenases, shares four absolutely conserved histidines that coordinate the iron required for activity of these enzymes (22,27). Because RPE65 retains these histidines, it might also bind catalytic iron to play a role in isomerization.…”