The P450-4 gene of Gibberella fujikuroi encodes ent-kaurene oxidase in the gibberellin biosynthesis pathway.
At least five genes of the gibberellin (GA) biosynthesis pathway are clustered on chromosome 4 of Gibberella fujikuroi; these genes encode the bifunctional ent-copalyl diphosphate synthase/ent-kaurene synthase, a GA-specific geranylgeranyl diphosphate synthase, and three cytochrome P450 monooxygenases. We now describe a fourth cytochrome P450 monooxygenase gene (P450-4). Gas chromatography-mass spectrometry analysis of extracts of mycelia and culture fluid of a P450-4 knockout mutant identified ent-kaurene as the only intermediate of the GA pathway. Incubations with radiolabeled precursors showed that the metabolism of ent-kaurene, ent-kaurenol, and ent-kaurenal was blocked in the transformants, whereas ent-kaurenoic acid was metabolized efficiently to GA(4). The GA-deficient mutant strain SG139, which lacks the 30-kb GA biosynthesis gene cluster, converted ent-kaurene to ent-kaurenoic acid after transformation with P450-4. The B1-41a mutant, described as blocked between ent-kaurenal and ent-kaurenoic acid, was fully complemented by P450-4. There is a single nucleotide difference between the sequence of the B1-41a and wild-type P450-4 alleles at the 3' consensus sequence of intron 2 in the mutant, resulting in reduced levels of active protein due to a splicing defect in the mutant. These data suggest that P450-4 encodes a multifunctional ent-kaurene oxidase catalyzing all three oxidation steps between ent-kaurene and ent-kaurenoic acid.
Tudzynski
B
B
Westfälische Wilhelms-Universität Münster, Institut für Botanik, Schlossgarten 3, D-48149 Münster, Germany. Bettina.Tudzynski@uni-muenster.de
Hedden
P
P
Carrera
E
E
Gaskin
P
P
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003470:Culture Media; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005874:Gibberella / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D005875:Gibberellins / Q000096:biosynthesis; D008969:Molecular Sequence Data; D009154:Mutation; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D014466:Ultraviolet Rays
D003470:Culture Media; D005875:Gibberellins; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases; C066398:ent-kaurene oxidase