Mutant B1-41a Research Articles

Biosynthetic Origin of Gibberellins A3 and A7 in Cell-Free Preparations from Seeds of Marah macrocarpus and Malus domestica

Cell-free preparations from seeds of Marah macrocarpus L. and Malus domestica L. catalyzed the conversion of gibberellin A(9) (GA(9)) and 2,3-dehydroGA(9) to GA(7); GA(9) was also metabolized to GA(4) in a branch pathway. The preparation from Marah seeds also metabolized GA(5) to GA(3) in high yield; GA(6) was a minor product and was not metabolized to GA(3). Using substrates stereospecifically labeled with deuterium, it was shown that the metabolism of GA(5) to GA(3) and of 2,3-dehydroGA(9) to GA(7) occurs with the loss of the 1beta-hydrogen. In cultures of Gibberella fujikuroi, mutant B1-41a, [1beta,2beta-(2)H(2)]GA(4), was metabolized to [1,2-(2)H(2)]GA(3) with the loss of the 1alpha- and 2alpha-hydrogens. These results provide further evidence that the biosynthetic origin of GA(3) and GA(7) in higher plants is different from that in the fungus Gibberella fujikuroi.

Stereochemistry of the metabolic steps from kaurenoic acids to kaurenolides and gibberellins

The metabolism of kaurenoic acid, labelled with deuterium at the 6α-, 6β-, 7α- and 7β-positions, has been examined in a microsomal enzyme system from the endosperm of Cucurbita maxima seeds and in cultures of Gibberella fujikuroi mutant B1-41a. The deuterium content in the products from each substrate was determined by capillary GC-MS. The results show that: (i) kaurenoic acid is metabolised to kaura-6,16-dienoic acid, and hence to the kaurenolides, by the stereospecific loss of the 7β-hydrogen and the non-stereospecific loss of the 6α- or 6β-hydrogen; (ii) 6β- and 7β- hydroxylation of kaurenoic acid occur with retention of configuration; (iii) the metabolism of kaurenoic acid to GA 12- aldehyde occurs with the loss of the 6β- and 7β-hydrogens and retention of the 6α- and 7α-hydrogens, the latter being lost from the 7-aldehyde on further metabolism to the gibberellins.

Gibberellins in seeds of Helianthus annuus

Fourteen gibberellins (GAs) have been identified in seeds of Helianthus annuus. They comprise the previously known GA 1, GA 4, GA 19, GA 20, and GA 45, and nine new 15β-hydroxyGAs. Of the new GAs, 15β-hydroxyGA 20 and 15β-hydroxyGA 1 have been previously synthesized chemically from GA 3 and are now assigned the numbers GA 67 and GA 72. The structures of 15β-hydroxyGA 15, 15β-hydroxyGA 24, and 15β-hydroxyGA 25 were established by their formation from the incubation of ent-15β-hydroxykaur-16-en-19-oic acid with cultures of Gibberella fujikuroi, mutant B1-41a; these new GAs have been respectively assigned the numbers, GA 64, GA 65 and GA 66. The remaining four GAs have been tentatively assigned the structures, 15β-hydroxyGA 17, 15β-hydroxyGA 19, 15β-hydroxyGA 44 and 15β-hydroxyGA 53 on the basis of the mass spectra of their MeTMSi derivatives. Abscisic acid, trans-abscisic acid, dihydrophaseic acid, dioxindole-3-acetic acid, ent-7α,16α,17-trihydroxy-16αH-kauranoic acid and ent-7α,16β,17-trihydroxy-and ent-6α,7α,16β,17-tetrahydroxy-16βH-kauranoic acids have also been detected in seeds of Helianthus annuus by capillary GC-MS. From the incubation of ent-15α-hydroxykaur-16-en-19-oic acid with Gibberella fujikuroi, mutant B1-41a, ent-7α,15α-dihydroxykaur-16-en-19-oic acid and 7β,15β-dihydroxykaurenolide have been isolated.

Microbiological conversion of 12-oxygenated and other derivatives of ent-kaur-16-en-19-oic acid by Gibberella Fujikuroi, mutant B1-41a

The metabolism of several ring C and D-functionalized ent-kaur-16-en-19-oic acids by cultures of Gibberella fujikuroi, mutant B1-41a, to the corresponding derivatives of the normal fungal gibberellins (GAs) and ent-kaurenoids is described. A range of 12α- and 12β-hydroxyGAs and ent-kaurenoids are characterized by their mass spectra and GC Kovats retention indices. The mass spectral and GC data are used to identify the 12α-hydroxy derivatives of GA 12, GA 14, GA 37 and GA 4 (GA 58), and of the 12β-hydroxy derivatives of ent-7α-hydroxy- and ent-6α, 7α-dihydroxykaurenoic acids, in seeds of Cucurbita maxima. Similarly the metabolites of GA 9, formed in seeds of Pisum sativum and cultures of G.fujikuroi, mutant B1-41a, are identified as 12α-hydroxyGA 9. ent-11β-Hydroxy- and ent-11-oxo-kaurenoic acids are metabolized by the fungus to the corresponding 11-oxygenated derivatives of the normal fungal ent-kaurenoids and some C 20-GAs; no 11-oxygenated C 19-GAs are formed. Grandiflorenic acid, 11β-hydroxygrandiflorenic acid, attractyligen and ent-15β-hydroxykaurenoic acid are metabolized to unidentified products.

Diterpene acids from Helianthus species and their microbiological conversion by Gibberella fujikuroi, mutant B1-41a

The diterpene acid content in 10 species of Helianthus has been investigated. Ent-12,16-cyclokauranoic acid, isolated from H. annuus, is converted into a series of 12,16-cyclogibberellins by cultures of Gibberella fujikuroi, mutant B1-41a, and 12,16-cyclogibberellins A 9, and A 12 have been isolated. Ent-12β-acetoxykaurenoic acid and ent-13( S)-angeloxyatisenoic acid have been isolated from H. decapetalus; the metabolism of ent-13( S)-hydroxyatisenoic acid and atisenoic acid by B1-41a is also described.

Mechanism and stereochemistry of conjugate reduction of enones from gibberellins A3 and A7

Conjugate reduction of the methyl esters of 3-didehydrogibberellin A3 13-acetate and of 3-didehydrogibberellin A7, in aprotic solvents by borohydride (or borodeuteride), is shown to introduce hydrogen (or deuterium) at the 1β- and 3β-positions in the products, 3-epi-gibberellin A1 13-acetate and 3-epi-gibberellin A4 methyl esters. The third hydrogen (or deuterium) comes from the proton (or deuteron) source used in the work-up. A mechanism for conjugate reduction of enones is proposed. The products from the borodeuteride reduction of 3-didehydrogibberellin A7 methyl ester with proton and deuteron work-up were chemically converted into [1β-2H1]-, [1β,22H2]-, and [1β3α-2H2]-gibberellin A4 and the stereochemistries of the deuterium atoms were determined from the deuterium content of the metabolites, formed from these labelled gibberellins in cultures of Gibberella fujikuroi, mutant B1-41a.

Conversion of trachylobanic acid into novel pentacyclic analogues of gibberellins by Gibberella fujikuroi, mutant B1-41a

12,16-Cyclogibborellins A9 and A12 have been isolated from the microbiological transformation of trachylobanic acid by Gibberella fujikuroi, mutant B1-41a, thus providing a notable example of the non-specificity of enzymes, catalysing the biosynthesis of fungal gibberellins; the 12,16-cyclogibberellin A9 retains gibberellin-like biological activity.

Fungal products. Part 20. Transformations of 2- and 3-hydroxylated kaurenoids by Gibberella fujikuroi

ent-3α-Hydroxy- and ent-2α,3α-dihydroxy-kaur-16-en-19-oic acids have been prepared from ent-kaur-16-ene-3β,19-diol, and their metabolites from cultures of the mutant B1-41a of Gibberella fujikuroi have been analysed by g.l.c.–mass spectrometry. The metabolism of ent-2α-hydroxy, ent-2β-hydroxy-, and ent-3α-hydroxykaur-16-en-19-ols has been similarly investigated in cultures of the parent wild-type strain, GF-1a, in which gibberellin biosynthesis was blocked by a synthetic plant growth retardant. The results show that the ent-3α-hydroxylated analogues of the normal gibberellin intermediates, ent-kaur-16-en-19-ol and ent-kaur-16-en-19-oic acid, are efficiently converted into 3-hydroxylated gibberellins. They also indicate that the ent-2β-hydroxy-analogue is converted into gibberellin A3 by dehydration and that the conversion of ent-kaurenoids into gibbereilins is reduced by the presence of ent-2α- and ent-2β-hydroxy-groups.

Fungal products. Part XVI. Conversion of isosteviol and steviol acetate into gibberellin analogues by mutant b1-41a of Gibberella fujikuroi and the preparation of (3H)gibberellin A20.

Isosteviol (ent-16-oxobeyeran-19-oic acid) and steviol acetate (ent-13-acetoxykaur-16-en-19-oic acid) are efficiently metabolised by cultures of resuspended mycelium of Gibberella fujikuroi, mutant B1–41a. Isosteviol is converted, inter alia, into ring-CD-rearranged derivatives of gibberellins A17, A19, A20, and 13-hydroxygibberellin A12. Steviol acetate is metabolised mainly to the 7β-hydroxy- and 6β, 7β-dihydroxy-derivatives and to the 13-acetates of gibberellins A17 and A20. The preparation of [3H]steviol acetate and its conversion into [3H]gibberellin A20 of high specific activity is described. These results provide further evidence that the fungal enzymes for gibberellin biosynthesis from ent-kaurenoic acid lack substrate specificity. They also reveal that structural changes in the CD ring system of ent-kaurenoid substrates suppress the 3-hydroxylating enzyme.

Fungal products. Part XVII. Microbiological hydroxylation of gibberellin A9 and its methyl ester

Gibberellin A9(1) is metabolised by re-suspended cultures of Gibberella fujikuroi, mutant B1-41a, principally to gibberellins A20(17) and A40(18). Other metabolites, detected by g.l.c.–mass spectrometry include gibberellin A10(2) and A11(24); two didehydrogibberellin A9 derivatives (12) and (14); 16α,17-dihydro-16,17-dihydroxy-gibberellin A9(26); 3-epi-gibberellin A4(4); and a monohydroxygibberellin A9, possibly (19).

The metabolism of steviol to 13-hydroxylated ent-Gibberellanes and ent-kauranes

Steviol( ent-13-hydroxykaur-16-en-19-oic acid) is rapidly metabolised by the mutant B1-41a of Gibberellafujikuroi. The initial product is the ent- 7-α-hydroxy derivative which is then further metabolised to gibberellins A 1, A 18, A 19, A 20, 13-hydroxy GA 12, the ent-6α, 7α, 13- and ent-6β, 7α, 13 (19,6-lactone)-trihydroxykaurenoic acids, and a seco-ring B diacid. This apparently low substrate specificity of the enzymes operative beyond the block in the mutant B1-41a provides a useful model for the biosynthetic pathways to 13-hydroxylated gibberellins of higher plants and a preparative route to these plant gibberellins.

Fungal products. Part XIV. Metabolic pathways from ent-kaurenoic acid to the fungal gibberellins in mutant B1-41a of Gibberella fujikuroi

The metabolism of substrates normally produced by wild-type strains of Gibberella fujikuroi has been determined in resuspended cultures of the mutant B1-41a at pH 3·5 and 7·0. The metabolites were identified by g.l.c.–mass spectrometry and g.l.c.–radio-counting. From the results at pH 3·5 the metabolic steps from ent-kaurenoic acid to the fungal gibberellins have been deduced. The oxidation level at which C-20 is lost in the formation of the C19-gibberellins could not be determined. ent-7-Oxokaurenoic acid does not serve as a precursor of gibberellin A12 aldehyde. At pH 7·0 the pathway from gibberellin A12 is diverted completely to 3-deoxygibberellins such as gibberellin A9.

Position of the metabolic block for gibberellin biosynthesis in mutant b1-41a of Gibberella fujikuroi

Mutant B1-41a, obtained by UV-irradiation of Gibberella fujikuroi strain GF-1a, does not metabolise mevalonic acid lactone (MVL), ent-kaur-16-ene, ent-kaurenol, and ent-kaurenal to gibberellins. ent-Kaur-16-ene-19-oic acid is completely metabolised to give the same gibberellins in similar concentration as unsupplemented cultures of the parent strain. It is concluded that this mutant is blocked for gibberellin synthesis at the step from ent-kaurenal to ent-kaurenoic acid. Comparison of the incorporation of MVL into GA 3 by the mutant and the parent strains indicate that the metabolic block is 97·5% effective. A method of preparing ent-kaur-16-ene, labelled at C-15 and C-17 by [ 2H] and [ 3H] is described.

Metabolism of steviol and its derivatives by Gibberella fujikuroi, mutant B1-41a

The low substrate specificity of enzymes operating beyond the genetic defect in mutant B1–41a of the fungus, Gibberella fujikuroi, is shown by the metabolism of the non-fungal diterpene steviol (ent-13-hydroxykaur-16-en-19-oic acid) and some of its derivatives to higher plant gibberellins and their derivatives.

Gibberellin biosynthesis in the mutant B1-41a of Gibberella fujikuroi

The mutant B1-41a of Gibberella fujikuroi is shown to be blocked for gibberellin synthesis at the step between ent-kaurenal and ent-kaurenoic acid; steps beyond the block have been examined by feeding, to the mutant, substrates which occur beyond this point in the parent strain GF-1a.