RESUMO
The biosynthesis of gibberellic acid (GA(3)) by the fungus Fusarium fujikuroi is catalyzed by seven enzymes encoded in a gene cluster. While four of these enzymes are characterized as cytochrome P450 monooxygenases, the nature of a fifth oxidase, GA(4) desaturase (DES), is unknown. DES converts GA(4) to GA(7) by the formation of a carbon-1,2 double bond in the penultimate step of the pathway. Here, we show by expression of the des complementary DNA in Escherichia coli that DES has the characteristics of a 2-oxoglutarate-dependent dioxygenase. Although it has low amino acid sequence homology with known 2-oxoglutarate-dependent dioxygenases, putative iron- and 2-oxoglutarate-binding residues, typical of such enzymes, are apparent in its primary sequence. A survey of sequence databases revealed that homologs of DES are widespread in the ascomycetes, although in most cases the homologs must participate in non-gibberellin (GA) pathways. Expression of des from the cauliflower mosaic virus 35S promoter in the plant species Solanum nigrum, Solanum dulcamara, and Nicotiana sylvestris resulted in substantial growth stimulation, with a 3-fold increase in height in S. dulcamara compared with controls. In S. nigrum, the height increase was accompanied by a 20-fold higher concentration of GA(3) in the growing shoots than in controls, although GA(1) content was reduced. Expression of des was also shown to partially restore growth in plants dwarfed by ectopic expression of a GA 2-oxidase (GA-deactivating) gene, consistent with GA(3) being protected from 2-oxidation. Thus, des has the potential to enable substantial growth increases, with practical implications, for example, in biomass production.
Assuntos
Proteínas Fúngicas/isolamento & purificação , Fusarium/enzimologia , Oxigenases de Função Mista/isolamento & purificação , Nicotiana/crescimento & desenvolvimento , Solanum/crescimento & desenvolvimento , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Caulimovirus/enzimologia , Caulimovirus/genética , Caulimovirus/metabolismo , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Bases de Dados Genéticas , Ensaios Enzimáticos/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fusarium/genética , Vetores Genéticos , Giberelinas/biossíntese , Giberelinas/genética , Giberelinas/metabolismo , Ácidos Cetoglutáricos/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Dados de Sequência Molecular , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Regiões Promotoras Genéticas , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Solanum/genética , Solanum/metabolismo , Especificidade por Substrato , Nicotiana/genética , Nicotiana/metabolismoRESUMO
Immature seeds of the southern wild cucumber, Marah macrocarpus, are a rich source of gibberellins (GAs) and were used in some of the earliest experiments on GA biosynthesis. The main biologically active GAs in developing embryos and endosperm of M. macrocarpus are GA(4) and GA(7), which have been shown previously to be formed from GA(9) in separate pathways, GA(4) being formed directly by 3ß-hydroxylation, while GA(7) is produced in two steps via 2,3-didehydroGA(9). In order to identify the enzymes responsible for these conversions, three cDNA clones encoding functionally different GA 3-oxidases, MmGA3ox1, -2 and -3, were obtained from young immature M. macrocarpus embryos. Their biochemical functions were determined by expression of the cDNAs in Escherichia coli and incubation of cell lysates with (14)C-labelled substrates. MmGA3ox1 and MmGA3ox3 converted GA(9) to GA(4) as sole product, while MmGA3ox2 produced several products, including GA(4), 2,3-didehydroGA(9), 2,3-epoxyGA(9), GA(20) and GA(5), these last two products requiring 13-hydroxylation of GA(9) and 2,3-didehydroGA(9), respectively. MmGA3ox1 converted 2,3-didehydroGA(9) to GA(7), while MmGA3ox3 converted this substrate to the 2,3-epoxide, and MmGA3ox2 also formed the epoxide, but also GA(5.) Thus, formation of GA(7) requires the sequential activities of MmGA3ox2 and MmGA3ox1, while MmGA3ox3 is not involved in GA(7) production. The enzymes catalysed similar reactions when incubated with 13-hydroxylated GAs, although with reduced efficiencies. The 13-hydroxylase activity of MmGA3ox2 may be responsible for the production of GA(1) and GA(3), which are present at low levels in developing M. macrocarpus seeds.
