Stereochemical inversion of (S)-reticuline by a cytochrome P450 fusion in opium poppy

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.

Structural and Functional Studies of Pavine N-Methyltransferase from Thalictrum flavum Reveal Novel Insights into Substrate Recognition and Catalytic Mechanism

“…in the structures of the apoenzyme as well as the complexes containing SAH or SAM). The steady-state kinetic parameters for THP appear similar to those for pavine (22), except for a significant amount of apparent substrate inhibition at higher concentrations of THP. The binding of (R)-THP in a non-productive binding mode next to (S)-THP, which is bound in a productive mode, suggests that the (R)-THP may inhibit the release of products from the enzyme when higher concentrations of the racemic substrate are added to the reaction.…”

“…Isolated THP was identified as a 1:1 racemic mixture of enantiomers using chiral chromatography ( Fig. 2A), following a previously published protocol (22).…”

Exploiting plant alkaloids

“…The necessity of that fusion protein for morphine production in planta was detected by the analysis of mutant poppy lines that lost the ability to produce morphinan alkaloids and instead accumulated (S)-reticuline and its metabolic derivatives. The same enzyme function was also proven by virus induced gene silencing (VIGS), which was initially aimed at suppression of codeinone reductase several steps downstream from reticuline epimerization ( Figure 2) [10]. However, (S)-reticuline accumulated instead of codeinone and indicated, which enzyme expression was hit by the VIGS experiment.…”

From Photodriven Charge Accumulation to Fueling Enzyme Cascades in Molecular Factories

“…[13] With the aim of developing molecular factories incorporating both natural and artificial enzymes, we have demonstrated that such artificial metalloenzymes based on the biotin-streptavidin technology i) maintain their activity upon incorporation within artificial organelles [14] and ii) are fully compatible with a broad palette or natural enzymes. [1] We propose to engineer compartmentalized molecular factories consisting of an artificial photosynthetic reaction center, an artificial imine reductase, [13] and an hemedependent monooxygenase [15] for the production of high added value alkaloids (Fig. 4).…”