Phytoalexin Synthesis in Soybean: Purification and Reconstitution of Cytochrome P450 3,9-dihydroxypterocarpan 6a-hydroxylase and Separation from Cytochrome P450 Cinnamate 4-hydroxylase

Elicitor-challenged soybean (Glycine max) cell cultures were used for detergent solubilization and purification of cytochrome P450 3,9-dihydroxypterocarpan 6a-hydroxylase (D6aH). D6aH was purified to electrophoretic homogeneity from such cells by a five-step procedure. It could be separated from cytochrome P450 cinnamate 4-hydroxylase on hydroxyapatite. This is the first report on separation of two cytochrome P450 enzymes from a higher plant. On sodium dodecyl sulfate polyacrylamide gels D6aH migrated with a Mr about 55,000. For reconstitution experiments soybean NADPH:cytochrome P450 (cytochrome c) reductase was purified to homogeneity. Reconstitution of D6aH in the presence of NADPH was dependent on cytochrome P450 D6aH, the reductase, and lipid. Dilauroylphosphatidylcholine gave higher D6aH activity than soybean lipids (asolectin). The reconstituted D6aH system showed a much higher temperature stability than the microsomal system.