Identification, expression, and substrate specificity of a mammalian beta-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 microm. 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.
Redmond
T M
TM
Laboratory of Retinal Cell and Molecular Biology, NEI, National Institutes of Health, Bethesda, Maryland 20892, USA. redmond@helix.nih.gov
Gentleman
S
S
Duncan
T
T
Yu
S
S
Wiggert
B
B
Gantt
E
E
Cunningham
F X
FX
D000595:Amino Acid Sequence; D000818:Animals; D002338:Carotenoids / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D018076:DNA, Complementary / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D010105:Oxygenases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D013379:Substrate Specificity; D014018:Tissue Distribution; D050559:beta-Carotene 15,15'-Monooxygenase
D018076:DNA, Complementary; D012333:RNA, Messenger; D002338:Carotenoids; D010105:Oxygenases; C493359:Bco1 protein, mouse; D050559:beta-Carotene 15,15'-Monooxygenase