title: Kaurenolides and fujenoic acids are side products of the gibberellin P450-1 monooxygenase in Gibberella fujikuroi.
abstract: The steps involved in kaurenolide and fujenoic acids biosynthesis, from ent-kauradienoic acid and ent-6alpha,7alpha-dihydroxykaurenoic acid, respectively, are demonstrated in the gibberellin (GA)-deficient Gibberella fujikuroi mutant SG139, which lacks the entire GA-biosynthesis gene cluster, complemented with the P450-1 gene of GA biosynthesis (SG139-P450-1). ent-[2H]Kauradienoic acid was efficiently converted into 7beta-hydroxy[2H]kaurenolide and 7beta,18-dihydroxy[2H]kaurenolide by the cultures while 7beta-hydroxy[2H]kaurenolide was transformed into 7beta,18-dihydroxy[2H]kaurenolide. The limiting step was found to be hydroxylation at C-18. In addition, SG139-P450-1 transformed ent-6alpha,7alpha-dihydroxy[14C4]kaurenoic acid into [14C4]fujenoic acid and [14C4]fujenoic triacid. Fujenal was also converted into the same products but was demonstrated not to be an intermediate in this sequence. All the above reactions were absent in the mutant SG139 and were suppressed in the wild-type strain ACC917 by disruption of the P450-1 gene. Kaurenolide and fujenoic acids synthesis were associated with the microsomal fraction and showed an absolute requirement for NADPH or NADH, all properties of cytochrome P450 monooxygenases. Only 7beta-hydroxy[14C4]kaurenolide synthesis and not further 18-hydroxylation was detected in the microsomal fraction. The substrates for the P450-1 monooxygenase, ent-kaurenoic acid and [2H]GA12, efficiently inhibited kaurenolide synthesis with I50 values of 3 and 6 microM, respectively. Both substrates also inhibited ent-6alpha,7alpha-dihydroxy[14C4]kaurenoic acid metabolism by SG139-P450-1. Conversely, [14C4]GA14 synthesis from [14C4]GA12-aldehyde was inhibited by ent-[2H]kauradienoic acid and fujenal with I50 values of 10 and 30 microM, respectively. These results demonstrate that kaurenolides and seco-ring B kaurenoids are formed by the P450-1 monooxygenase (GA14 synthase) of G. fujikuroi and are thus side products that probably result from stabilization of radical intermediates involved in GA14 synthesis.
authors.affiliation: Departamento de Química, Laboratorio de Bioorgánica, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile. crojas@uchile.cl
authors.forename: M Cecilia
authors.initials: MC
authors.lastname: Rojas
authors.forename: Oriana
authors.initials: O
authors.lastname: Urrutia
authors.forename: Carlos
authors.initials: C
authors.lastname: Cruz
authors.forename: Paul
authors.initials: P
authors.lastname: Gaskin
authors.forename: Bettina
authors.initials: B
authors.lastname: Tudzynski
authors.forename: Peter
authors.initials: P
authors.lastname: Hedden
mesh_terms: D000447:Aldehydes / Q000737:chemistry / Q000378:metabolism*; D002250:Carbon Radioisotopes; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004224:Diterpenes / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005874:Gibberella / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D005875:Gibberellins / Q000096:biosynthesis*; D007527:Isoenzymes; D008861:Microsomes / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D013379:Substrate Specificity; D014170:Transformation, Genetic
chemical_list: D000447:Aldehydes; D002250:Carbon Radioisotopes; D065607:Cytochrome P-450 Enzyme Inhibitors; D004224:Diterpenes; D004791:Enzyme Inhibitors; D005875:Gibberellins; D007527:Isoenzymes; C452220:kauradienoic acid; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; C051436:kaurenoic acid; D003577:Cytochrome P-450 Enzyme System