Molecular characterization and functional expression of dihydroxypterocarpan 6a-hydroxylase, an enzyme specific for pterocarpanoid phytoalexin biosynthesis in soybean (Glycine max L.).
Four cytochrome P450-dependent enzymes, among them dihydroxypterocarpan 6a-hydroxylase (D6aH), are specifically involved in the elicitor-inducible biosynthesis of glyceollins, the phytoalexins of soybean. Here we report that CYP93A1 cDNA, which we isolated previously from elicitor-induced soybean cells, codes for a protein with D6aH activity. Analysis of the catalytic properties of recombinant CYP93A1 expressed in yeast, its NADPH dependency, stereoselectivity and high substrate affinity confirmed that D6aH is the physiological function of CYP93A1. It thus represents the first isoflavonoid-specific CYP to be characterized at the molecular level. In elicitor-treated soybean cells producing phytoalexins, increases in D6aH activity were correlated with elevated transcript levels which indicates that expression of the enzyme is regulated at the level of transcription. Therefore, CYP93A1 cDNA can be used as a specific molecular marker for the inducible defense response against pathogen attack.
Botanisches Institut, Ludwig-Maximilians-Universität München, Munich, Germany.
C R
CR
Schopfer
G
G
Kochs
F
F
Lottspeich
J
J
Ebel
D000595:Amino Acid Sequence; D001578:Benzopyrans / Q000378:metabolism*; D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000187:drug effects / Q000235:genetics*; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D005936:Glucans / Q000494:pharmacology; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000187:drug effects / Q000235:genetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry; D010936:Plant Extracts / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics*; D017434:Protein Structure, Tertiary; D036343:Pterocarpans; D011994:Recombinant Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D012717:Sesquiterpenes; D030262:Soybean Proteins; D013025:Glycine max / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics*; D013045:Species Specificity; D013379:Substrate Specificity; D013729:Terpenes / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics; D000094162:Phytoalexins
D001578:Benzopyrans; D005936:Glucans; D009842:Oligopeptides; D010936:Plant Extracts; D010940:Plant Proteins; D036343:Pterocarpans; D011994:Recombinant Proteins; D012717:Sesquiterpenes; D030262:Soybean Proteins; D013729:Terpenes; C017343:glyceollin; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C042062:cytochrome P450 CYP93A1 (Glycine max); D000094162:Phytoalexins