The biochemical characterization of ferret carotene‐9′,10′‐monooxygenase catalyzing cleavage of carotenoids in vitro

We have previously demonstrated apo‐10′‐lycopenoid formation from lycopene cleavage by ferret carotene‐9′,10′‐monooxygenase (CMO) expressed in Cos‐1 cells and in ferrets (a model for human carotenoid metabolism) supplemented with lycopene in vivo , suggesting the oxidative cleavage of lycopene at its 9′,10′ double bond. In this report, we provide further information on the biochemical characterization of ferret CMO, in terms of the kinetic analysis for β‐carotene/lycopene cleavage into apo‐10′‐carotenoid/apo‐10′‐lycopenoid in vitro . The recombinant CMO of ferret was expressed from baculovirus‐infected Spodoptera frugiperda 9 insect cells and was confirmed by immunoblotting with a purified polyclonal antibody against N‐terminal amino acids of ferret CMO with the predicted molecular weight of 60 kDa. The kinetic analysis of ferret CMO showed that the CMO effectively cleaved both all‐trans β‐carotene and 5‐ cis and 13‐ cis ‐isomers of lycopene at the 9′,10′ double bond to produce β‐apo‐10′‐carotenal and apo‐10′‐lycopenal, respectively, in a time‐, pH‐, protein‐ and substrate dose‐dependant manner. Only trace amounts of apo‐10′‐lycopenal were detected when all‐trans lycopene was incubated as substrate. The Km of ferret CMO for β‐carotene and lycopene isomers (containing ~20% as cis isomers) was 3.5±1.1 mM and 4.6±1.1 mM, respectively. Iron, but not NAD or DTT, was an essential co‐factor for the reaction. In summary, these data suggest that ferret CMO preferentially cleaves cis ‐isomers of lycopene into apo‐10′‐lycopenal, but not all‐trans lycopene. Our finding of preferential bioconversion of cis ‐isomers of lycopene into apo‐10′‐lycopenoids by CMO is significant because cis ‐isomers of lycopene are a predominant form of lycopene in mammalian tissue. Supported by the NIH CA104932 and USDA 1950‐51000‐064.