RESUMO
The gateway to morphine biosynthesis in opium poppy (Papaver somniferum) is the stereochemical inversion of (S)-reticuline since the enzyme yielding the first committed intermediate salutaridine is specific for (R)-reticuline. A fusion between a cytochrome P450 (CYP) and an aldo-keto reductase (AKR) catalyzes the S-to-R epimerization of reticuline via 1,2-dehydroreticuline. The reticuline epimerase (REPI) fusion was detected in opium poppy and in Papaver bracteatum, which accumulates thebaine. In contrast, orthologs encoding independent CYP and AKR enzymes catalyzing the respective synthesis and reduction of 1,2-dehydroreticuline were isolated from Papaver rhoeas, which does not accumulate morphinan alkaloids. An ancestral relationship between these enzymes is supported by a conservation of introns in the gene fusions and independent orthologs. Suppression of REPI transcripts using virus-induced gene silencing in opium poppy reduced levels of (R)-reticuline and morphinan alkaloids and increased the overall abundance of (S)-reticuline and its O-methylated derivatives. Discovery of REPI completes the isolation of genes responsible for known steps of morphine biosynthesis.
Assuntos
Aldeído Redutase/metabolismo , Carboidratos Epimerases/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação da Expressão Gênica de Plantas , Morfina/biossíntese , Papaver/metabolismo , Proteínas de Plantas/metabolismo , Aldeído Redutase/genética , Aldo-Ceto Redutases , Alcaloides/biossíntese , Alcaloides/química , Sequência de Bases , Benzilisoquinolinas/química , Benzilisoquinolinas/metabolismo , Bromoviridae/genética , Bromoviridae/metabolismo , Carboidratos Epimerases/antagonistas & inibidores , Carboidratos Epimerases/genética , Sistema Enzimático do Citocromo P-450/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Éxons , Fusão Gênica , Íntrons , Ligases/genética , Ligases/metabolismo , Dados de Sequência Molecular , Morfinanos/química , Morfinanos/metabolismo , Morfina/química , Fases de Leitura Aberta , Ópio/química , Ópio/metabolismo , Oxirredução , Papaver/genética , Proteínas de Plantas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , EstereoisomerismoRESUMO
Many molecular phylogenies of viruses build upon the analysis of single genes. The study of whole-genomes, however, might yield more reliable information to infer tree topologies and a better approach for drawing the evolutionary history of virus families. In this study, we apply a novel comparative proteomic approach to seek whether incorporating information from the entire proteome would support the actual taxonomy of the family Bromoviridae. Our results suggest that the current taxonomic classification should be modified in several aspects to account for the genomic properties of the Bromoviridae. These differences are: i) Alfalfa mosaic virus (AMV) is a true Ilarvirus instead of constituting an independent genus; ii) Pelargonium zonate spot virus (PZSV) should be considered as a member of the Bromoviridae; and iii) the genus Ilarvirus should be divided into fewer phylogenetic subgroups than suggested by antigenic differences.
Assuntos
Bromoviridae/classificação , Bromoviridae/metabolismo , Evolução Molecular , Filogenia , Bromoviridae/química , Bromoviridae/genética , Perfilação da Expressão Gênica , Proteoma/genética , Proteoma/metabolismo , ProteômicaRESUMO
The RNA binding properties of the prunus necrotic ringspot virus (PNRSV) coat protein (CP) were demonstrated by northwestern and dot-blot analyses. The capability to bind PNRSV RNA 4 was compared with viruses representing three different interactions prevailing in the assembly and architecture of virions. The results showed that cucumber mosaic virus (CMV) and PNRSV CPs, which stabilise their virions mainly through RNA-protein interactions bound PNRSV RNA 4 even at very high salt concentrations. The CP of cherry leaf roll nepovirus, whose virions are predominantly stabilised by protein-protein interactions did not bind even at the lowest salt concentration tested. Finally the CP of carnation mottle carmovirus, that has an intermediate position in which both RNA-protein and protein-protein interactions are equally important showed a salt-dependent RNA binding.