apocarotenal and alpha-carotene

Provitamin A carotenoids are oxidatively cleaved by β-carotene 15,15'-dioxygenase (BCO1) at the central 15-15' double bond to form retinal (vitamin A aldehyde). Another carotenoid oxygenase, β-carotene 9',10'-oxygenase (BCO2) catalyzes the oxidative cleavage of carotenoids at the 9'-10' bond to yield an ionone and an apo-10'-carotenoid. Previously published substrate specificity studies of BCO2 were conducted using crude lysates from bacteria or insect cells expressing recombinant BCO2. Our attempts to obtain active recombinant human BCO2 expressed in Escherichia coli were unsuccessful. We have expressed recombinant chicken BCO2 in the strain E. coli BL21-Gold (DE3) and purified the enzyme by cobalt ion affinity chromatography. Like BCO1, purified recombinant chicken BCO2 catalyzes the oxidative cleavage of the provitamin A carotenoids β-carotene, α-carotene, and β-cryptoxanthin. Its catalytic activity with β-carotene as substrate is at least 10-fold lower than that of BCO1. In further contrast to BCO1, purified recombinant chicken BCO2 also catalyzes the oxidative cleavage of 9-cis-β-carotene and the non-provitamin A carotenoids zeaxanthin and lutein, and is inactive with all-trans-lycopene and β-apocarotenoids. Apo-10'-carotenoids were detected as enzymatic products by HPLC, and the identities were confirmed by LC-MS. Small amounts of 3-hydroxy-β-apo-8'-carotenal were also consistently detected in BCO2-β-cryptoxanthin reaction mixtures. With the exception of this activity with β-cryptoxanthin, BCO2 cleaves specifically at the 9'-10' bond to produce apo-10'-carotenoids. BCO2 has been shown to function in preventing the excessive accumulation of carotenoids, and its broad substrate specificity is consistent with this.

Topics: Amino Acid Sequence; Animals; beta Carotene; Carotenoids; Chickens; Cryptoxanthins; Dioxygenases; Humans; Recombinant Proteins; Sequence Alignment; Substrate Specificity

The Lys residue at position 108 of human beta-carotene 15,15'-monooxygenase is located on the outside surface of the active tunnel of the enzyme. Hydrophobic mutations (K108F and K108L) at this position substantially decreased the affinity of the enzyme for substrates with ionone rings at both ends, such as alpha-carotene, beta-carotene, and beta-cryptoxanthine. In contrast, these mutations had little effect on the affinity of the enzyme for substrates with one ionone ring and one open-chain end, such as beta-apo-4'-carotenal and beta-apo-8'-carotenal. The residue 108 may be related to the indirect interaction with the second ionone ring of the substrates with two ionone rings.

Topics: Amino Acid Sequence; beta Carotene; beta-Carotene 15,15'-Monooxygenase; Carotenoids; Cryptoxanthins; Humans; Hydrophobic and Hydrophilic Interactions; Lysine; Models, Molecular; Molecular Sequence Data; Norisoprenoids; Protein Binding; Sequence Alignment; Substrate Specificity; Xanthophylls