Engineering of a Water-Soluble Plant Cytochrome P450, CYP73A1, and NMR-Based Orientation of Natural and Alternate Substrates in the Active Site

Abstract

CYP73A1 catalyzes cinnamic acid hydroxylation, a reaction essential for the synthesis of lignin monomers and most phenolic compounds in higher plants. The native CYP73A1, initially isolated from Jerusalem artichoke (Helianthus tuberosus), was engineered to simplify purification from recombinant yeast and improve solublity and stability in the absence of detergent by replacing the hydrophobic N terminus with the peptitergent amphipathic sequence PD1. Optimized expression and purification procedures yielded 4 mg engineered CYP73A1 L-1 yeast culture. This water-soluble enzyme was suitable for 1H-nuclear magnetic resonance (NMR) investigation of substrate positioning in the active site. The metabolism and interaction with the enzyme of cinnamate and four analogs were compared by UV-visible and 1H-NMR analysis. It was shown that trans-3-thienylacrylic acid, trans-2-thienylacrylic acid, and 4-vinylbenzoic acid are good ligands and substrates, whereas trans-4-fluorocinnamate is a competitive inhibitor. Paramagnetic relaxation effects of CYP73A1-Fe(III) on the 1H-NMR spectra of cinnamate and analogs indicate that their average initial orientation in the active site is parallel to the heme. Initial orientation and distances of ring protons to the iron do not explain the selective hydroxylation of cinnamate in the 4-position or the formation of single products from the thienyl compounds. Position adjustments are thus likely to occur during the later steps of the catalytic cycle.