Substrate specificity of purified recombinant human ?-carotene 15,15'-oxygenase (BCO1).

ABSTRACT: Humans cannot synthesize vitamin A and thus must obtain it from their diet. ?-Carotene 15,15'-oxygenase (BCO1) catalyzes the oxidative cleavage of provitamin A carotenoids at the central 15-15' double bond to yield retinal (vitamin A). In this work, we quantitatively describe the substrate specificity of purified recombinant human BCO1 in terms of catalytic efficiency values (kcat/Km). The full-length open reading frame of human BCO1 was cloned into the pET-28b expression vector with a C-terminal polyhistidine tag, and the protein was expressed in the Escherichia coli strain BL21-Gold(DE3). The enzyme was purified using cobalt ion affinity chromatography. The purified enzyme preparation catalyzed the oxidative cleavage of ?-carotene with a Vmax = 197.2 nmol retinal/mg BCO1 × h, Km = 17.2 ?M and catalytic efficiency kcat/Km = 6098 M(-1) min(-1). The enzyme also catalyzed the oxidative cleavage of ?-carotene, ?-cryptoxanthin, and ?-apo-8'-carotenal to yield retinal. The catalytic efficiency values of these substrates are lower than that of ?-carotene. Surprisingly, BCO1 catalyzed the oxidative cleavage of lycopene to yield acycloretinal with a catalytic efficiency similar to that of ?-carotene. The shorter ?-apocarotenals (?-apo-10'-carotenal, ?-apo-12'-carotenal, ?-apo-14'-carotenal) do not show Michaelis-Menten behavior under the conditions tested. We did not detect any activity with lutein, zeaxanthin, and 9-cis-?-carotene. Our results show that BCO1 favors full-length provitamin A carotenoids as substrates, with the notable exception of lycopene. Lycopene has previously been reported to be unreactive with BCO1, and our findings warrant a fresh look at acycloretinal and its alcohol and acid forms as metabolites of lycopene in future studies.

SUBMITTER: dela Sena C 

PROVIDER: S-EPMC3873565 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature