RESUMO
Momilactones are bioactive diterpenoids that contribute to plant defense against pathogens and allelopathic interactions between plants. Both cultivated and wild grass species of Oryza and Echinochloa crus-galli (barnyard grass) produce momilactones using a biosynthetic gene cluster (BGC) in their genomes. The bryophyte Calohypnum plumiforme (formerly Hypnum plumaeforme) also produces momilactones, and the bifunctional diterpene cyclase gene CpDTC1/HpDTC1, which is responsible for the production of the diterpene framework, has been characterized. To understand the molecular architecture of the momilactone biosynthetic genes in the moss genome and their evolutionary relationships with other momilactone-producing plants, we sequenced and annotated the C. plumiforme genome. The data revealed a 150-kb genomic region that contains two cytochrome P450 genes, the CpDTC1/HpDTC1 gene and the "dehydrogenase momilactone A synthase" gene tandemly arranged and inductively transcribed following stress exposure. The predicted enzymatic functions in yeast and recombinant assay and the successful pathway reconstitution in Nicotiana benthamiana suggest that it is a functional BGC responsible for momilactone production. Furthermore, in a survey of genomic sequences of a broad range of plant species, we found that momilactone BGC is limited to the two grasses (Oryza and Echinochloa) and C. plumiforme, with no synteny among these genomes. These results indicate that while the gene cluster in C. plumiforme is functionally similar to that in rice and barnyard grass, it is likely a product of convergent evolution. To the best of our knowledge, this report of a BGC for a specialized plant defense metabolite in bryophytes is unique.
Assuntos
Evolução Molecular , Genoma de Planta , Lactonas/metabolismo , Plantas/metabolismo , Vias Biossintéticas , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/classificação , Plantas/genéticaRESUMO
The biosynthesis of isopentenyl diphosphate, a fundamental precursor for isoprenoids, via the mevalonate pathway is completed by diphosphomevalonate decarboxylase. This enzyme catalyzes the formation of isopentenyl diphosphate through the ATP-dependent phosphorylation of the 3-hydroxyl group of (R)-5-diphosphomevalonate followed by decarboxylation coupled with the elimination of the 3-phosphate group. In this reaction, a conserved aspartate residue has been proposed to be involved in the phosphorylation step as the general base catalyst that abstracts a proton from the 3-hydroxyl group. In this study, the catalytic mechanism of this rare type of decarboxylase is re-investigated by structural and mutagenic studies on the enzyme from a thermoacidophilic archaeon Sulfolobus solfataricus The crystal structures of the archaeal enzyme in complex with (R)-5-diphosphomevalonate and adenosine 5'-O-(3-thio)triphosphate or with (R)-5-diphosphomevalonate and ADP are newly solved, and theoretical analysis based on the structure suggests the inability of proton abstraction by the conserved aspartate residue, Asp-281. Site-directed mutagenesis on Asp-281 creates mutants that only show diphosphomevalonate 3-kinase activity, demonstrating that the residue is required in the process of phosphate elimination/decarboxylation, rather than in the preceding phosphorylation step. These results enable discussion of the catalytic roles of the aspartate residue and provide clear proof of the involvement of a long predicted intermediate, (R)-3-phospho-5-diphosphomevalonate, in the reaction of the enzyme.
Assuntos
Substituição de Aminoácidos , Carboxiliases/química , Fosfotransferases/química , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Conformação Proteica , Espectrometria de Massas por Ionização por Electrospray , Especificidade por Substrato , Sulfolobus solfataricus/enzimologiaRESUMO
The lack of a few conserved enzymes in the classical mevalonate pathway and the widespread existence of isopentenyl phosphate kinase suggest the presence of a partly modified mevalonate pathway in most archaea and in some bacteria. In the pathway, (R)-mevalonate 5-phosphate is thought to be metabolized to isopentenyl diphosphate via isopentenyl phosphate. The long anticipated enzyme that catalyzes the reaction from (R)-mevalonate 5-phosphate to isopentenyl phosphate was recently identified in a Cloroflexi bacterium, Roseiflexus castenholzii, and in a halophilic archaeon, Haloferax volcanii. However, our trial to convert the intermediates of the classical and modified mevalonate pathways into isopentenyl diphosphate using cell-free extract from a thermophilic archaeon Thermoplasma acidophilum implied that the branch point intermediate of these known pathways, i.e. (R)-mevalonate 5-phosphate, is unlikely to be the precursor of isoprenoid. Through the process of characterizing the recombinant homologs of mevalonate pathway-related enzymes from the archaeon, a distant homolog of diphosphomevalonate decarboxylase was found to catalyze the phosphorylation of (R)-mevalonate to yield (R)-mevalonate 3-phosphate. The product could be converted into isopentenyl phosphate, probably through (R)-mevalonate 3,5-bisphosphate, by the action of unidentified T. acidophilum enzymes fractionated by anion-exchange chromatography. These findings demonstrate the presence of a third alternative "Thermoplasma-type" mevalonate pathway, which involves (R)-mevalonate 3-phosphotransferase and probably both (R)-mevalonate 3-phosphate 5-phosphotransferase and (R)-mevalonate 3,5-bisphosphate decarboxylase, in addition to isopentenyl phosphate kinase.
Assuntos
Ácido Mevalônico/análogos & derivados , Ácido Mevalônico/metabolismo , Thermoplasma/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Sequência de Bases , Sistema Livre de Células , Cromatografia por Troca Iônica , Cromatografia em Camada Fina , Primers do DNA , FilogeniaRESUMO
The hormonal action of jasmonate in plants is controlled by the precise balance between its biosynthesis and inactivation. Oxidation of jasmonoyl-L-isoleucine at the C-12 position, which is catalyzed by cytochrome P450s CYP94B3 and CYP94C1, is thought to be one of the main inactivation pathways. In this study, an additional function of CYP94B3 was elucidated, as well additional jasmonoyl-L-isoleucine metabolites being investigated. It was found that CYP94B3 also catalyzes the hydroxylation of jasmonoyl-L-valine and jasmonoyl-L-phenylalanine, and that these hydroxyl compounds accumulated after wounding and possessed lower activity than non-hydroxylated compounds. Additionally, 12-O-ß-glucopyranosyl-jasmonoyl-L-isoleucine accumulated after wounding, suggesting that it is a metabolite of jasmonoyl-L-isoleucine.
Assuntos
Ciclopentanos/química , Ciclopentanos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Glucosídeos/metabolismo , Isoleucina/análogos & derivados , Oxilipinas/química , Oxilipinas/metabolismo , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Biocatálise , Glucosídeos/química , Isoleucina/química , Isoleucina/metabolismo , Conformação Molecular , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Glycine max/metabolismo , Nicotiana/metabolismoRESUMO
Polyphosphate kinase (PPK), which can regenerate ATP from ADP, was utilized in the mevalonate-dependent enzymatic synthesis of amorphadiene. The activity of PPK, cloned from Escherichia coli, was determined by (31)P-NMR. The yield from the PPK-catalyzed synthesis was 25%, 2.5 times higher than that without PPK. The (31)P-NMR analysis of the final reaction mixture indicated no accumulation of intermediates.
Assuntos
Trifosfato de Adenosina/química , Proteínas de Bactérias/química , Ácido Mevalônico/química , Fosfotransferases (Aceptor do Grupo Fosfato)/química , Sesquiterpenos/síntese química , Proteínas de Bactérias/genética , Biocatálise , Clonagem Molecular , Escherichia coli/genética , Espectroscopia de Ressonância Magnética , Fosfotransferases (Aceptor do Grupo Fosfato)/genética , Sesquiterpenos Policíclicos , Polifosfatos/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , SoluçõesRESUMO
Brassinolide (BL), a plant 7-oxalactone-type steroid hormone, is one of the active brassinosteroids (BRs) that regulates plant growth and development. BL is biosynthesized from castasterone by the cytochrome P450 monooxygenase, CYP85A2. We showed that a Pichia pastoris transformant that synchronously expresses Arabidopsis P450 reductase gene ATR1 and P450 gene CYP85A2 converts teasterone and typhasterol to 7-oxateasterone and 7-oxatyphasterol, respectively. Thus, CYP85A2 catalyzes the lactonization reactions of not only castasterone but also teasterone and typhasterol. The two 2-deoxy-7-oxalactone-type BRs were identified in Arabidopsis plants. Although the reversible conversion between 7-oxateasterone and 7-oxatyphasterol was observed in vivo, no conversion of 7-oxatyphasterol to BL was observed. The biological activity of 7-oxatyphasterol toward Arabidopsis hypocotyl elongation was nearly the same as that of castasterone. These results suggest that a new BR biosynthetic pathway, a BR lactonization pathway, functions in Arabidopsis and plays an important role in regulating the concentration of active BRs, even though the metabolism of 7-oxatyphasterol to BL is still unknown.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Sistema Enzimático do Citocromo P-450/metabolismo , Lactonas/química , Esteroides/biossíntese , Esteroides/química , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Catálise , Sistema Enzimático do Citocromo P-450/genética , Cromatografia Gasosa-Espectrometria de Massas , Estrutura Molecular , Pichia/genética , Pichia/metabolismo , Proteínas Proto-Oncogênicas c-myb/genética , Proteínas Proto-Oncogênicas c-myb/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transgenes/genéticaRESUMO
The structure of a germination inhibitor, hypnosin, isolated from phytopathogenic Streptomyces sp. causing root tumor of melon was determined to be 3-acetylaminopyrazine-2-carboxylic acid (1) by mass spectrometry, computational chemical prediction of UV spectrum, and synthesis of candidates. The structure-activity relationship of hypnosin and anthranilic acid was examined, and it was concluded that pyrazinecarboxylic acid or pyridine-2-carboxylic acid was the fundamental structure with activity, that methylation of the carboxyl group or decarboxylation destroyed activity, and that the presence of an amino group was inhibitory to the activity, whereas acetylation or deletion of an amino group enhanced activity. Hypnosin inhibited spore germination of some Streptomyces spp. in addition to the species with which it was isolated.
Assuntos
Cucumis/microbiologia , Doenças das Plantas/microbiologia , Pirazinas/química , Esporos Bacterianos/efeitos dos fármacos , Esporos Bacterianos/crescimento & desenvolvimento , Streptomyces/crescimento & desenvolvimento , Ácidos Carboxílicos/química , Ácidos Carboxílicos/farmacologia , Temperatura Alta , Pirazinas/farmacologia , Piridinas/química , Streptomyces/química , Streptomyces/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
The germination rate and activation conditions of spores were examined for four strains of Streptomyces sp., a phytopathogen causing root tumor of melon. An inhibitor was isolated from the agar-cultured material of strain CB-1-1 and then characterized. The inhibitor selectively acted on spore germination and did not affect hyphal growth, and inhibition was abolished by washing the spores in water. The inhibitor was produced by an agar culture, and most of the inhibitor existed in the spores. The IC(50) value for the inhibitor was approximately 0.25 microg/ml.
Assuntos
Doenças das Plantas/microbiologia , Esporos Fúngicos/efeitos dos fármacos , Streptomyces/metabolismo , Fenômenos Químicos , Físico-Química , Cucurbitaceae/microbiologia , Meios de Cultura , Temperatura Alta , Hifas/efeitos dos fármacos , Hifas/crescimento & desenvolvimento , Espectroscopia de Ressonância Magnética , Dodecilsulfato de Sódio/farmacologia , Espectrometria de Massas por Ionização por Electrospray , Espectrofotometria Ultravioleta , Esporos Fúngicos/crescimento & desenvolvimento , Streptomyces/crescimento & desenvolvimento , Tensoativos/farmacologiaRESUMO
ent-Kaurene is the key intermediate in biosynthesis of gibberellins (GAs), plant hormones. In higher plants, ent-kaurene is synthesized successively by copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS) from geranylgeranyl diphosphate (GGDP). On the other hand, fungal ent-kaurene synthases are bifunctional cyclases with both CPS and KS activity in a single polypeptide. The moss Physcomitrella patens is a model organism for the study of genetics and development in an early land plant. We identified ent-kaurene synthase (PpCPS/KS) from P. patens and analyzed its function. PpCPS/KS cDNA encodes a 101-kDa polypeptide, and shows high similarity with CPSs and abietadiene synthase from higher plants. PpCPS/KS is a bifunctional cyclase and, like fungal CPS/KS, directly synthesizes the ent-kaurene skeleton from GGDP. PpCPS/KS has two aspartate-rich DVDD and DDYFD motifs observed in CPS and KS, respectively. The mutational analysis of two conserved motifs in PpCPS/KS indicated that the DVDD motif is responsible for CPS activity (GGDP to CDP) and the DDYFD motif for KS activity (CDP to ent-kaurene and ent-16alpha-hydroxykaurene).