Escherichia coli as a platform for functional expression of plant P450 carotene hydroxylases

Abstract

Carotenoids and their derivatives are essential for growth, development, and signaling in plants and have an added benefit as nutraceuticals in food crops. Despite the importance of the biosynthetic pathway, there remain open questions regarding some of the later enzymes in the pathway. The CYP97 family of P450 enzymes was predicted to function in carotene ring hydroxylation, to convert provitamin A carotenes to nonprovitamin A xanthophylls. However, substrate specificity was difficult to investigate directly in plants, which mask enzyme activities by a complex and dynamic metabolic network. To characterize the enzymes more directly, we amplified cDNAs from a model crop, Oryza sativa, and used functional complementation in Escherichia coli to test activity and specificity of members of Clans A and C. This heterologous system will be valuable for further study of enzyme interactions and substrate utilization needed to understand better the role of CYP97 hydroxylases in plant carotenoid biosynthesis.

Variabilidade de genótipos de milho quanto à composição de carotenoides nos grãos

“…Os analisados está na mesma rota metabólica (Dellapenna & Pogson, 2006;Quinlan et al, 2007). Houve forte correlação das frações carotenos, xantofi las monohidroxiladas e xantofi las di-hidroxiladas com os carotenoides totais (Tabela 3).…”

Synergistic Interactions between Carotene Ring Hydroxylases Drive Lutein Formation in Plant Carotenoid Biosynthesis

“…From genetic and functional complementation studies Kim and DellaPenna, 2006;Quinlan et al, 2007), it is generally accepted that CYP97C is an «-ring hydroxylase and CYP97A is a b-ring hydroxylase. Mutant phenotypes suggested that CYP97A hydroxylates the b-rings of a-carotene to form zeinoxanthin, which is the preferred substrate for «-ring hydroxylation by CYP97C (Kim and DellaPenna, 2006).…”

“…A widely used functional complementation method employed in our previous studies demonstrated activity of the P450 and HYD carotene ring hydroxylases from grasses (Quinlan et al, 2007;Vallabhaneni et al, 2009). Compared with b-ring hydroxylation by HYD, CYP97A4 and CYP97C2 were less effective in hydroxylating carotene rings in E. coli accumulating b-carotene or «-«-carotene, their respective b-ring or «-ring substrates.…”

“…We expressed rice (Oryza sativa) CYP97A4 and CYP97C2 (Quinlan et al, 2007) and maize (Zea mays) HYD4 (Vallabhaneni et al, 2009) in E. coli that accumulated both a-carotene (b-«-rings) and b-carotene (b-b-rings). Carotenoid products were analyzed by HPLC and/or liquid chromatography-mass spectrometry (LC-MS).…”

“…The pathway likely functions as a multienzyme complex(es) to facilitate metabolite channeling, as predicted by the absence of pathway intermediates and the presence of complexes containing carotenoid biosynthetic enzymes (Maudinas et al, 1977;Camara et al, 1982;Kreuz et al, 1982;AlBabili et al, 1996;Bonk et al, 1997;Lopez et al, 2008). In this study, we examined an intriguing portion of the pathway, where hydroxylation of rings in carotenes is catalyzed by two structurally distinct enzymes, P450 heme (CYP97) and nonheme diiron (HYD) enzymes DellaPenna, 2001, 2004;Kim and DellaPenna, 2006;Quinlan et al, 2007). Hydroxylation of the two b-ionone rings in b-carotene leads to a formation of zeaxanthin, while hydroxylation of the one b-ring and one «-ring in a-carotene leads to lutein.…”