ABSTRACT
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.
Subject(s)
Aldehyde Reductase/metabolism , Carbohydrate Epimerases/metabolism , Cytochrome P-450 Enzyme System/metabolism , Gene Expression Regulation, Plant , Morphine/biosynthesis , Papaver/metabolism , Plant Proteins/metabolism , Aldehyde Reductase/genetics , Aldo-Keto Reductases , Alkaloids/biosynthesis , Alkaloids/chemistry , Base Sequence , Benzylisoquinolines/chemistry , Benzylisoquinolines/metabolism , Bromoviridae/genetics , Bromoviridae/metabolism , Carbohydrate Epimerases/antagonists & inhibitors , Carbohydrate Epimerases/genetics , Cytochrome P-450 Enzyme System/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Exons , Gene Fusion , Introns , Ligases/genetics , Ligases/metabolism , Molecular Sequence Data , Morphinans/chemistry , Morphinans/metabolism , Morphine/chemistry , Open Reading Frames , Opium/chemistry , Opium/metabolism , Oxidation-Reduction , Papaver/genetics , Plant Proteins/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , StereoisomerismABSTRACT
OBJECTIVE: This study examines if homeopathic drug Arsenicum Album 30C (Ars Alb 30C) can elicit ameliorative responses in yeast (Saccharomyces cerevisiae) exposed to arsenate. METHODS: The yeast S. cerevisiae 699 was cultured in a standard yeast extract peptone dextrose broth medium. It was exposed to the final concentration of 0.15 mmol/L arsenate for two intervals, 1 h and 2 h, respectively. The cell viability was determined along with the assessment of several toxicity biomarkers such as catalase (CAT), superoxide dismutase (SOD), total thiol (GSH) and glucose-6-phosphate dehydrogenase (G6PDH), lipid peroxidation, protein carbonylation and DNA damage. Reactive oxygen species (ROS) accumulation, expressions of relevant stress transcription activators like Yap-1 and Msn 2, and mRNA expression of yeast caspase-1 (Yca-1) were also measured. RESULTS: Treatment of arsenate increased lipid peroxidation, protein carbonylation, DNA damage, ROS accumulation and expressions of Yap-1, Msn 2 and Yca-1 and decreased GSH, G6PDH, CAT and SOD. Ars Alb 30C administration decreased lipid peroxidation, protein carbonylation, DNA damage, ROS formation and Msn 2 and Yca-1 expressions and increased cell viability, GSH, G6PDH, CAT and SOD significantly (P<0.05), except for a slight increase in Yap-1 expression. CONCLUSION: Ars Alb 30C triggers ameliorative responses in S. cerevisiae exposed to arsenate.