Expression and characterization of a murine enzyme able to cleave beta-carotene. The formation of retinoids

• Published in: The Journal of biological chemistry 2001-08, Vol.276 (34), p.32160-32168
• Main Authors: Paik, J, During, A, Harrison, E H, Mendelsohn, C L, Lai, K, Blaner, W S
• Format: Article
• Language: English
• Subjects: Animals; b-carotene; b-carotene 15,15'-dioxygenase; beta Carotene - metabolism; beta-Carotene 15,15'-Monooxygenase; Blotting, Northern; Blotting, Western; CHO Cells; Chromatography, High Pressure Liquid; Cloning, Molecular; Cricetinae; DNA, Complementary; Escherichia coli; Humans; Hydrolysis; Kidney - enzymology; Mice; Oxygenases - chemistry; Oxygenases - genetics; Oxygenases - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; retinal; Retinoids - metabolism; Sequence Homology, Amino Acid
• ISSN: 0021-9258
• DOI: 10.1074/jbc.m010086200

Because animals are not able to synthesize retinoids de novo, ultimately they must derive them from dietary provitamin A carotenoids through a process known as carotene cleavage. The enzyme responsible for catalyzing carotene cleavage (beta-carotene 15,15'-dioxygenase) has been characterized primarily in rat intestinal scrapings. Using a recently reported cDNA sequence for a carotene cleavage enzyme from Drosophila, we identified a cDNA encoding a mouse homolog of this enzyme. When the cDNA was expressed in either Escherichia coli or Chinese hamster ovary cells, expression conferred upon bacterial and Chinese hamster ovary cell homogenates the ability to cleave beta-carotene to retinal. Several lines of evidence obtained upon kinetic analyses of the recombinant enzyme suggested that carotene cleavage enzyme interacts with other proteins present within cell or tissue homogenates. This was confirmed by pull-down experiments upon incubation of recombinant enzyme with tissue 12,000 x g supernatants. Matrix-assisted laser desorption ionization-mass spectrometry analysis of pulled-down proteins indicates that an atypical testis-specific isoform of lactate dehydrogenase associates with recombinant carotene cleavage enzyme. mRNA transcripts for the carotene cleavage enzyme were detected by reverse transcription-polymerase chain reaction in mouse testes, liver, kidney, and intestine. In situ hybridization studies demonstrated that carotene cleavage enzyme is expressed prominently in maternal tissue surrounding the embryo but not in embryonic tissues at 7.5 and 8.5 days postcoitus. This work offers new insights for understanding the biochemistry of carotene cleavage to retinoids.