Further characterization of cytochrome P450 involved in phytoalexin synthesis in soybean: Cytochrome P450 cinnamate 4-hydroxylase and 3,9-dihydroxypterocarpan 6a-hydroxylase

Abstract

Two cytochrome P450 enzymes, cinnamate 4-hydroxylase (C4H) and 3,9-dihydroxypterocarpan 6a-hydroxylase (D6aH), were isolated from elicitor-challenged soybean ( Glycine max) cell cultures ( G. Kochs and H. Grisebach, 1989, Arch. Biochem. Biophys. 273, 543–553 ). An earlier purification protocol was improved by the use of new chromatographic media, leading to a higher yield of enzymatic activity. After separation of C4H from D6aH on hydroxyapatite, the C4H was identified using anti-C4H antibody from Jerusalem artichoke ( Helianthus tuberosus) ( B. Gabriac et al., 1991, Arch. Biochem. Biophys. 288, 302–309 ). The two proteins show molecular weights of about 58,000 for C4H and about 55,000 for D6aH on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both enzyme activities are dependent on NADPH:cytochrome P450 reductase and cross-react with their respective antibodies. Both cytochrome P450 subspecies show substrate binding and CO-difference spectra typical for cytochrome P450 and were found to be glycoproteins by their cross-reaction with biotinylated lectins in Western blot. The N-terminal sequence of C4H from soybean shows high similarity to the N-terminus of C4H from Jerusalem artichoke.