Identification, expression, and substrate specificity of a mammalian β-carotene 15,15′-dioxygenase

We have identified from mouse the first mammalian beta -carotene 15,15'-dioxygenase (beta -CD), a crucial enzyme in development and metabolism that governs the de novo entry of vitamin A from plant-derived precursors. beta -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. beta -CD expression in Escherichia coli cells engineered to produce beta -carotene led to the accumulation of all-trans-retinal at the expense of beta -carotene, confirming that beta -CD catalyzed the central cleavage of this vitamin A precursor. Purified recombinant beta -CD protein cleaves beta -carotene in vitro with a V-max of 36 pmol of retinal/mg of enzyme/ min and a K-m of 6 muM. 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 beta -CD in an accessible model organism will create new opportunities to study vitamin A metabolism.