Cloning and Expression of β,β-Carotene 15,15′-Dioxygenase

• Published in: Biochemical and biophysical research communications 2000-05, Vol.271 (2), p.334-336
• Main Authors: Wyss, A., Wirtz, G., Woggon, W.-D., Brugger, R., Wyss, M., Friedlein, A., Bachmann, H., Hunziker, W.
• Format: Article
• Language: English
• Subjects: Animals; b,b-carotene 15,15'-dioxygenase; beta-Carotene 15,15'-Monooxygenase; Cell Line; centric cleavage; Chickens; Chromatography, High Pressure Liquid; Cloning, Molecular; Cricetinae; Cytoplasm - enzymology; DNA, Complementary - metabolism; duodenal expression library; Duodenum - enzymology; Electrophoresis, Polyacrylamide Gel; Oxygenases - chemistry; Oxygenases - genetics; Oxygenases - metabolism; Peptides - chemistry; Recombinant Proteins; retinal; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; vitamin A; β,β-carotene 15,15′-dioxygenase; β-carotene
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1006/bbrc.2000.2619

β,β-Carotene 15,15′-dioxygenase cleaves β-carotene into two molecules of retinal and is therefore the key enzyme in β-carotene metabolism to vitamin A. In the present study, it was possible to enrich the chicken β,β-carotene 15,15′-dioxygenase to such an extent that partial amino acid sequence information could be obtained to design degenerate oligonucleotides. With RT-PCR a cDNA fragment could be obtained and used subsequently in a radioactive screening of a chicken duodenal expression library. We cloned the first eukaryotic β,β-carotene 15,15′-dioxygenase which symmetrically cleaves β-carotene at the 15,15′-double bond.