In vitro characterization of a recombinant Blh protein from an uncultured marine bacterium as a beta-carotene 15,15'-dioxygenase.
Codon optimization was used to synthesize the blh gene from the uncultured marine bacterium 66A03 for expression in Escherichia coli. The expressed enzyme cleaved beta-carotene at its central double bond (15,15') to yield two molecules of all-trans-retinal. The molecular mass of the native purified enzyme was approximately 64 kDa as a dimer of 32-kDa subunits. The K(m), k(cat), and k(cat)/K(m) values for beta-carotene as substrate were 37 mum, 3.6 min(-1), and 97 mm(-1) min(-1), respectively. The enzyme exhibited the highest activity for beta-carotene, followed by beta-cryptoxanthin, beta-apo-4'-carotenal, alpha-carotene, and gamma-carotene in decreasing order, but not for beta-apo-8'-carotenal, beta-apo-12'-carotenal, lutein, zeaxanthin, or lycopene, suggesting that the presence of one unsubstituted beta-ionone ring in a substrate with a molecular weight greater than C(35) seems to be essential for enzyme activity. The oxygen atom of retinal originated not from water but from molecular oxygen, suggesting that the enzyme was a beta-carotene 15,15'-dioxygenase. Although the Blh protein and beta-carotene 15,15'-monooxygenases catalyzed the same biochemical reaction, the Blh protein was unrelated to the mammalian beta-carotene 15,15'-monooxygenases as assessed by their different properties, including DNA and amino acid sequences, molecular weight, form of association, reaction mechanism, kinetic properties, and substrate specificity. This is the first report of in vitro characterization of a bacterial beta-carotene-cleaving enzyme.
Kim
Yeong-Su
YS
Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, South Korea.
Kim
Nam-Hee
NH
Yeom
Soo-Jin
SJ
Kim
Seon-Won
SW
Oh
Deok-Kun
DK
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology*; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005822:Genetic Vectors; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D015183:Restriction Mapping; D012172:Retinaldehyde / Q000378:metabolism; D012623:Seawater / Q000382:microbiology*; D013379:Substrate Specificity; D019207:beta Carotene / Q000378:metabolism; D050559:beta-Carotene 15,15'-Monooxygenase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D019207:beta Carotene; D050559:beta-Carotene 15,15'-Monooxygenase; D012172:Retinaldehyde