Poster abstracts

Poster number 49 submitted by Carlo dela Sena

Biochemical Characterization and Substrate Specificity of Purified Recobinant Human β-Carotene 15-15′-Oxygenase 1 (BCO1)

Carlo C. dela Sena (Ohio State Biochemistry Program), Earl H. Harrison (Department of Human Nutrition, The Ohio State University)

Abstract:
Beta-carotene 15-15′-oxygenase 1 (BCO1) cleaves provitamin A carotenoids at the 15-15′ bond to produce vitamin A. Most BCO1 substrate specificity studies were done by incubating crude or partially purified cell homogenates with test compounds (reviewed by Lietz et al.¹). Here we report the expression and purification of recombinant human BCO1 in Escherichia coli. The full length open reading frame of human BCMO1 was cloned into the pET-28b expression vector with a C-terminal polyhistidine tag, and the protein was expressed using BL21-Gold(DE3). The enzyme was purified using cobalt ion affinity chromatography. The purified enzyme has a catalytic efficiency (k_cat/K_M) of 1.92 x 10⁶ M^-1min^-1 using β-carotene as substrate. This is higher than the catalytic efficiencies of purified BCO1 preparations reported previously^2-3, which are in the order of 10⁵. No eccentric cleavage products (products produced from cleavages other than the 15-15′ bond) were detected. In agreement with the previous substrate specificity studies, our BCO1 preparation cleaved β-cryptoxanthin, α-carotene, β-apo-8′-carotenal, and β-apo-10′-carotenal, but neither lutein nor zeaxanthin. The higher catalytic efficiency of our BCO1 preparation also enabled us to detect cleavage activity on lycopene and β-apo-12′-carotenal, which were previously reported to be unreactive with BCO1^2-3.

References:
(1) Lietz, G.; Lange, J.; Rimbach, G. Arch Biochem Biophys 2010, 502, 8.
(2) Lindqvist, A.; Andersson, S. J Biol Chem 2002, 277, 23942.
(3) Kim, Y. S.; Oh, D. K. Biotechnol Lett 2009, 31, 403.

Keywords: Carotenoids, vitamin A, BCO1