Characterization, Purification, and Reconstitution of an Inducible Cytochrome P450-Dependent Triasulfuron Hydroxylase from Wheat

Abstract

This report describes the further purification and characterization as well as the solubilization and reconstitution of an inducible cytochrome P450 monooxygenase responsible for triasulfuron hydroxylation in wheat. Improved procedures for increasing the levels of expressed microsomal cytochrome P450 and triasulfuron hydroxylase activity in etiolated wheat seedling shoot tissues are described. Cytochrome P450 concentrations and triasulfuron hydroxylase activities were increased to 1.17 nmol mg−1 protein and 2.87 nmol min−1 mg−1 protein, respectively, with an improved enzyme assay system and the use of sucrose density gradient-purified microsomal preparations from naphthalic anhydride- and ethanol-induced coleoptile tissues. A pH optimum between 7.0 and 7.5 in potassium phosphate buffer and an apparent Km of 24 μM for triasulfuron were determined. Dependence of triasulfuron hydroxylation on cytochrome P450 was established with cytochrome P450 inhibitors (tetcyclacis, paclobutrazol, and piperonyl butoxide), reduced CO difference spectra, light-reversible CO inhibition, type I substrate and type II inhibitor binding spectra, and by inhibition with polyclonal antibodies raised against a partially purified fraction of wheat NADPH cytochrome c reductase (EC 1.6.2.4). Microsomal NADPH cytochrome c reductase and cytochrome P450 were solubilized with detergent (1% Chaps, v/v), separated by affinity chromatography on 2′,5′ADP-Sepharose 4B, and purified by DEAE ion exchange chromatography. Triasulfuron hydroxylase activity was reconstituted by chromatography of the purified NADPH cytochrome c reductase and cytochrome P450 fractions on hydroxyapatite. In preliminary studies with the reconstituted enzyme system, triasulfuron hydroxylase activity was 6% of the induced microsomal activity.