Production and structural characterization of the cytochrome P450 enzymes in carotene ring hydroxylation

Abstract

The superfamily of cytochrome P450 (CYP) enzymes participate in diverse metabolic reactions. Although structural characterization of prokaryotic CYPs has been performed extensively, structural studies for eukaryotic, especially plant, CYPs remain scanty. Members of CYP family 97 are hydroxylases devoted to carotene ring hydroxylation. Specifically, members of the CYP97A and CYP97C subfamilies work synergistically to hydroxylate the β- and ɛ-rings of α-carotene to form lutein. The stereospecific reactions introduce an R-chiral center at the C-3 position of each cyclohexene ring. The Arabidopsis thaliana genome encodes CYP97A3, CYP97B3, and CYP97C1. Recently, we determined crystal structures of CYP97A3 in unliganded and retinaldehyde-bound forms and of CYP97C1 in complex with detergent octylthioglucoside. These structures showed how the active site favors pro-R-hydroxylation. This chapter summarizes the steps for production of CYP97A3, CYP97B3, and CYP97C1. The in vitro assays to test hydroxylase activity and ligand binding affinity are also described, including the steps for production of the redox partner. We also review the details for crystallization and structural determination of CYP97A3 and CYP97C1. Characterization of the active site by high-resolution structure analysis provides clues to substrate channeling, which awaits further investigation.

Keywords: Crystallography; Cytochrome P450; FNR; Ferredoxin; Heterologous expression; Lutein.