Further characterization of cytochrome P450 involved in phytoalexin synthesis in soybean: cytochrome P450 cinnamate 4-hydroxylase and 3,9-dihydroxypterocarpan 6a-hydroxylase.
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.
Kochs
G
G
Biologisches Institut II, Universität, Lehrstuhl für Biochemie der Pflanzen, Freiburg, Germany.
Werck-Reichhart
D
D
Grisebach
H
H
D015153:Blotting, Western; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism*; D006031:Glycosylation; D006899:Mixed Function Oxygenases / Q000302:isolation & purification / Q000378:metabolism*; D010936:Plant Extracts / Q000096:biosynthesis*; D012717:Sesquiterpenes; D030262:Soybean Proteins; D013025:Glycine max / Q000201:enzymology*; D013057:Spectrum Analysis; D013729:Terpenes; D050564:Trans-Cinnamate 4-Monooxygenase; D000094162:Phytoalexins
D010936:Plant Extracts; D012717:Sesquiterpenes; D030262:Soybean Proteins; D013729:Terpenes; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C042062:cytochrome P450 CYP93A1 (Glycine max); D050564:Trans-Cinnamate 4-Monooxygenase; D000094162:Phytoalexins