The P450-4 gene of Gibberella fujikuroi encodes ent-kaurene oxidase in the gibberellin biosynthesis pathway

Appl Environ Microbiol. 2001 Aug;67(8):3514-22. doi: 10.1128/AEM.67.8.3514-3522.2001.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Cloning, Molecular
  • Culture Media
  • Cytochrome P-450 Enzyme System / chemistry
  • Cytochrome P-450 Enzyme System / genetics*
  • Cytochrome P-450 Enzyme System / metabolism*
  • Gibberella / enzymology*
  • Gibberella / genetics
  • Gibberella / growth & development
  • Gibberellins / biosynthesis
  • Molecular Sequence Data
  • Mutation
  • Oxygenases / chemistry
  • Oxygenases / genetics*
  • Oxygenases / metabolism*
  • Sequence Alignment
  • Sequence Analysis, DNA
  • Ultraviolet Rays


  • Culture Media
  • Gibberellins
  • Cytochrome P-450 Enzyme System
  • Oxygenases
  • ent-kaurene oxidase

Associated data

  • GENBANK/Y17243