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

At least five genes of the gibberellin (GA) biosynthesis pathway are cluste red on chromosome 4 of Gibberella fujikuroi; these genes encode the bifunct ional ent-copalyl diphosphate synthase/ent-kaurene synthase, a GA-specific geranylgeranyl diphosphate synthase, and three cytochrome P450 monooxygenas es. We now describe a fourth cytochrome P450 monooxygenase gene (P450-4). G as chromatography-mass spectrometry analysis of extracts of mycelia and cul ture fluid of a P450-4 knockout mutant identified ent-kaurene as the only i ntermediate of the GA pathway. Incubations with radiolabeled precursors sho wed that the metabolism of ent-kaurene, ent-kaurenol, and ent-kaurenal was blocked in the transformants, whereas ent-kaurenoic acid was metabolized ef ficiently to GA(4). The GA-deficient mutant strain SG139, which lacks the 3 0-kb GA biosynthesis gene cluster, converted ent-kaurene to ent-kaurenoic a cid after transformation with P450-4. The B1-41a mutant, described as block ed between ent-kaurenal and ent-kaurenoic acid, was fully complemented by P 450-4. 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 spl icing defect in the mutant. These data suggest that P450-4 encodes a multif unctional ent-kaurene oxidase catalyzing all three oxidation steps between ent-kaurene and ent-kaurenoic acid.