A cane toad (Rhinella marina) N-methyltransferase converts primary indolethylamines to tertiary psychedelic amines.

Psychedelic indolethylamines have emerged as potential medicines to treat several psychiatric pathologies. Natural sources of these compounds include 'magic mushrooms' (Psilocybe spp.), plants used to prepare ayahuasca, and toads. The skin and parotid glands of certain toads accumulate a variety of specialized metabolites including toxic guanidine alkaloids, lipophilic alkaloids, poisonous steroids, and hallucinogenic indolethylamines such as DMT, 5-methoxy-DMT, and bufotenin. The occurrence of psychedelics has contributed to the ceremonial use of toads, particularly among Mesoamerican peoples. Yet, the biosynthesis of psychedelic alkaloids has not been elucidated. Herein, we report a novel indolethylamine N-methyltransferase (RmNMT) from cane toad (Rhinella marina). The RmNMT sequence was used to identify a related NMT from the common toad, Bufo bufo. Close homologs from various frog species were inactive, suggesting a role for psychedelic indolethylamine biosynthesis in toads. Enzyme kinetic analyses and comparison with functionally similar enzymes showed that recombinant RmNMT was an effective catalyst and not product inhibited. The substrate promiscuity of RmNMT enabled the bioproduction of a variety of substituted indolethylamines at levels sufficient for purification, pharmacological screening, and metabolic stability assays. Since the therapeutic potential of psychedelics has been linked to activity at serotonergic receptors, we evaluated binding of derivatives at 5-HT1A and 5-HT2A receptors. Primary amines exhibited enhanced affinity at the 5-HT1A receptor compared with tertiary amines. With the exception of 6-substituted derivatives, N,N-dimethylation also protected against catabolism by liver microsomes.

Elucidation of the mescaline biosynthetic pathway in peyote (Lophophora williamsii).

Peyote (Lophophora williamsii) is an entheogenic and medicinal cactus native to the Chihuahuan desert. The psychoactive and hallucinogenic properties of peyote are principally attributed to the phenethylamine derivative mescaline. Despite the isolation of mescaline from peyote over 120 years ago, the biosynthetic pathway in the plant has remained undiscovered. Here, we use a transcriptomics and homology-guided gene discovery strategy to elucidate a near-complete biosynthetic pathway from l-tyrosine to mescaline. We identified a cytochrome P450 that catalyzes the 3-hydroxylation of l-tyrosine to l-DOPA, a tyrosine/DOPA decarboxylase yielding dopamine, and four substrate-specific and regiospecific substituted phenethylamine O-methyltransferases. Biochemical assays with recombinant enzymes or functional analyses performed by feeding putative precursors to engineered yeast (Saccharomyces cerevisiae) strains expressing candidate peyote biosynthetic genes were used to determine substrate specificity, which served as the basis for pathway elucidation. Additionally, an N-methyltransferase displaying broad substrate specificity and leading to the production of N-methylated phenethylamine derivatives was identified, which could also function as an early step in the biosynthesis of tetrahydroisoquinoline alkaloids in peyote.

Structural studies of codeinone reductase reveal novel insights into aldo-keto reductase function in benzylisoquinoline alkaloid biosynthesis.

Benzylisoquinoline alkaloids (BIAs) are a class of specialized metabolites with a diverse range of chemical structures and physiological effects. Codeine and morphine are two closely related BIAs with particularly useful analgesic properties. The aldo-keto reductase (AKR) codeinone reductase (COR) catalyzes the final and penultimate steps in the biosynthesis of codeine and morphine, respectively, in opium poppy (Papaver somniferum). However, the structural determinants that mediate substrate recognition and catalysis are not well defined. Here, we describe the crystal structure of apo-COR determined to a resolution of 2.4 Å by molecular replacement using chalcone reductase as a search model. Structural comparisons of COR to closely related plant AKRs and more distantly related homologues reveal a novel conformation in the ß1α1 loop adjacent to the BIA-binding pocket. The proximity of this loop to several highly conserved active-site residues and the expected location of the nicotinamide ring of the NADP(H) cofactor suggest a model for BIA recognition that implies roles for several key residues. Using site-directed mutagenesis, we show that substitutions at Met-28 and His-120 of COR lead to changes in AKR activity for the major and minor substrates codeinone and neopinone, respectively. Our findings provide a framework for understanding the molecular basis of substrate recognition in COR and the closely related 1,2-dehydroreticuline reductase responsible for the second half of a stereochemical inversion that initiates the morphine biosynthesis pathway.

Isolation and characterization of two O-methyltransferases involved in benzylisoquinoline alkaloid biosynthesis in sacred lotus (Nelumbo nucifera).

Benzylisoquinoline alkaloids (BIAs) are a major class of plant metabolites with many pharmacological benefits. Sacred lotus (Nelumbo nucifera) is an ancient aquatic plant of medicinal value because of antiviral and immunomodulatory activities linked to its constituent BIAs. Although more than 30 BIAs belonging to the 1-benzylisoquinoline, aporphine, and bisbenzylisoquinoline structural subclasses and displaying a predominant R-enantiomeric conformation have been isolated from N. nucifera, its BIA biosynthetic genes and enzymes remain unknown. Herein, we report the isolation and biochemical characterization of two O-methyltransferases (OMTs) involved in BIA biosynthesis in sacred lotus. Five homologous genes, designated NnOMT1-5 and encoding polypeptides sharing >40% amino acid sequence identity, were expressed in Escherichia coli Functional characterization of the purified recombinant proteins revealed that NnOMT1 is a regiospecific 1-benzylisoquinoline 6-O-methyltransferase (6OMT) accepting both R- and S-substrates, whereas NnOMT5 is mainly a 7-O-methyltransferase (7OMT), with relatively minor 6OMT activity and a strong stereospecific preference for S-enantiomers. Available aporphines were not accepted as substrates by either enzyme, suggesting that O-methylation precedes BIA formation from 1-benzylisoquinoline intermediates. Km values for NnOMT1 and NnOMT5 were 20 and 13 µm for (R,S)-norcoclaurine and (S)-N-methylcoclaurine, respectively, similar to those for OMTs from other BIA-producing plants. Organ-based correlations of alkaloid content, OMT activity in crude extracts, and OMT gene expression supported physiological roles for NnOMT1 and NnOMT5 in BIA metabolism, occurring primarily in young leaves and embryos of sacred lotus. In summary, our work identifies two OMTs involved in BIA metabolism in the medicinal plant N. nucifera.

PR10/Bet v1-like Proteins as Novel Contributors to Plant Biochemical Diversity.

Pathogenesis-related (PR) proteins constitute a broad class of plant proteins with analogues found throughout nature from bacteria to higher eukaryotes. PR proteins were first noted in plants as part of the hypersensitive response, but have since been assigned an array of biological roles. The PR10/Bet v1-like proteins are a subset of PR proteins characterized by an ability to bind a wide range of lipophilic ligands, uniquely positioning them as contributors to specialized biosynthetic pathways. PR10/Bet v1-like proteins participate in the production of plant alkaloids and phenolics including flavonoids, both as general binding proteins and in special cases as catalysts. Owing initially to the perceived allergenic properties of PR10/Bet v1-like proteins, many were studied at the structural level to elucidate the basis for ligand binding. These studies provided a foundation for more recent efforts to understand higher-level structural order and how PR10/Bet v1-like proteins catalyse key reactions in plant pathways. Synthetic biology aimed at reconstituting plant-specialized metabolism in microorganisms uses knowledge of these proteins to fine-tune performance in new systems.

Neopinone isomerase is involved in codeine and morphine biosynthesis in opium poppy.

The isomerization of neopinone to codeinone is a critical step in the biosynthesis of opiate alkaloids in opium poppy. Previously assumed to be spontaneous, the process is in fact catalyzed enzymatically by neopinone isomerase (NISO). Without NISO the primary metabolic products in the plant, in engineered microbes and in vitro are neopine and neomorphine, which are structural isomers of codeine and morphine, respectively. Inclusion of NISO in yeast strains engineered to convert thebaine to natural or semisynthetic opiates dramatically enhances formation of the desired products at the expense of neopine and neomorphine accumulation. Along with thebaine synthase, NISO is the second member of the pathogenesis-related 10 (PR10) protein family recently implicated in the enzymatic catalysis of a presumed spontaneous conversion in morphine biosynthesis.

Structure-function studies of tetrahydroprotoberberine N-methyltransferase reveal the molecular basis of stereoselective substrate recognition.

Benzylisoquinoline alkaloids (BIAs) are a structurally diverse class of plant-specialized metabolites that have been particularly well-studied in the order Ranunculales. The N-methyltransferases (NMTs) in BIA biosynthesis can be divided into three groups according to substrate specificity and amino acid sequence. Here, we report the first crystal structures of enzyme complexes from the tetrahydroprotoberberine NMT (TNMT) subclass, specifically for GfTNMT from the yellow horned poppy (Glaucium flavum). GfTNMT was co-crystallized with the cofactor S-adenosyl-l-methionine (dmin = 1.6 Å), the product S-adenosyl-l-homocysteine (dmin = 1.8 Å), or in complex with S-adenosyl-l-homocysteine and (S)-cis-N-methylstylopine (dmin = 1.8 Å). These structures reveal for the first time how a mostly hydrophobic L-shaped substrate recognition pocket selects for the (S)-cis configuration of the two central six-membered rings in protoberberine BIA compounds. Mutagenesis studies confirm and functionally define the roles of several highly-conserved residues within and near the GfTNMT-active site. The substrate specificity of TNMT enzymes appears to arise from the arrangement of subgroup-specific stereospecific recognition elements relative to catalytic elements that are more widely-conserved among all BIA NMTs. The binding mode of protoberberine compounds to GfTNMT appears to be similar to coclaurine NMT, with the isoquinoline rings buried deepest in the binding pocket. This binding mode differs from that of pavine NMT, in which the benzyl ring is bound more deeply than the isoquinoline rings. The insights into substrate recognition and catalysis provided here form a sound basis for the rational engineering of NMT enzymes for chemoenzymatic synthesis and metabolic engineering.

Purine Permease-Type Benzylisoquinoline Alkaloid Transporters in Opium Poppy.

Although opiate biosynthesis has been largely elucidated, and cell-to-cell transport has been long postulated, benzylisoquinoline alkaloid (BIA) transporters from opium poppy (Papaver somniferum) have not been reported. Investigation of a purine permease-type sequence within a recently discovered opiate biosynthetic gene cluster led to the discovery of a family of nine homologs designated as BIA uptake permeases (BUPs). Initial expression studies in engineered yeast hosting segments of the opiate pathway showed that six of the nine BUP homologs facilitated dramatic increases in alkaloid yields. Closer examination revealed the ability to uptake a variety of BIAs and certain pathway precursors (e.g. dopamine), with each BUP displaying a unique substrate acceptance profile. Improvements in uptake for yeast expressing specific BUPs versus those devoid of the heterologous transporters were high for early intermediates (300- and 25-fold for dopamine and norcoclaurine, respectively), central pathway metabolites [10-fold for (S)-reticuline], and end products (30-fold for codeine). A coculture of three yeast strains, each harboring a different consecutive segment of the opiate pathway and BUP1, was able to convert exogenous Levodopa to 3 ± 4 mg/L codeine via a 14-step bioconversion process involving over a dozen enzymes. BUP1 is highly expressed in opium poppy latex and is localized to the plasma membrane. The discovery of the BUP transporter family expands the role of purine permease-type transporters in specialized metabolism, and provides key insight into the cellular mechanisms involved in opiate alkaloid biosynthesis in opium poppy.

A pathogenesis-related 10 protein catalyzes the final step in thebaine biosynthesis.

The ultimate step in the formation of thebaine, a pentacyclic opiate alkaloid readily converted to the narcotic analgesics codeine and morphine in the opium poppy, has long been presumed to be a spontaneous reaction. We have detected and purified a novel enzyme from opium poppy latex that is capable of the efficient formation of thebaine from (7S)-salutaridinol 7-O-acetate at the expense of labile hydroxylated byproducts, which are preferentially produced by spontaneous allylic elimination. Remarkably, thebaine synthase (THS), a member of the pathogenesis-related 10 protein (PR10) superfamily, is encoded within a novel gene cluster in the opium poppy genome that also includes genes encoding the four biosynthetic enzymes immediately upstream. THS is a missing component that is crucial to the development of fermentation-based opiate production and dramatically improves thebaine yield in engineered yeast.

Back to the plant: overcoming roadblocks to the microbial production of pharmaceutically important plant natural products.

Microbial fermentation platforms offer a cost-effective and sustainable alternative to plant cultivation and chemical synthesis for the production of many plant-derived pharmaceuticals. Plant alkaloids, particularly benzylisoquinoline alkaloids and monoterpene indole alkaloids, and recently cannabinoids have become attractive targets for microbial biosynthesis owing to their medicinal importance. Recent advances in the discovery of pathway components, together with the application of synthetic biology tools, have facilitated the assembly of plant alkaloid and cannabinoid pathways in the microbial hosts Escherichia coli and Saccharomyces cerevisiae. This review highlights key aspects of these pathways in the framework of overcoming bottlenecks in microbial production to further improve end-product titers. We discuss the opportunities that emerge from a better understanding of the pathway components by further study of the plant, and strategies for generation of new and advanced medicinal compounds.

An N-methyltransferase from Ephedra sinica catalyzing the formation of ephedrine and pseudoephedrine enables microbial phenylalkylamine production.

Phenylalkylamines, such as the plant compounds ephedrine and pseudoephedrine and the animal neurotransmitters dopamine and adrenaline, compose a large class of natural and synthetic molecules with important physiological functions and pharmaceutically valuable bioactivities. The final steps of ephedrine and pseudoephedrine biosynthesis in members of the plant genus Ephedra involve N-methylation of norephedrine and norpseudoephedrine, respectively. Here, using a plant transcriptome screen, we report the isolation and characterization of an N-methyltransferase (NMT) from Ephedra sinica able to catalyze the formation of (pseudo)ephedrine and other naturally occurring phenylalkylamines, including N-methylcathinone and N-methyl(pseudo)ephedrine. Phenylalkylamine N-methyltransferase (PaNMT) shares substantial amino acid sequence identity with enzymes of the NMT family involved in benzylisoquinoline alkaloid (BIA) metabolism in members of the higher plant order Ranunculales, which includes opium poppy (Papaver somniferum). PaNMT accepted a broad range of substrates with phenylalkylamine, tryptamine, ß-carboline, tetrahydroisoquinoline, and BIA structural scaffolds, which is in contrast to the specificity for BIA substrates of NMT enzymes within the Ranunculales. PaNMT transcript levels were highest in young shoots of E. sinica, which corresponded to the location of NMT activity yielding (pseudo)ephedrine, N-methylcathinone, and N-methyl(pseudo)ephedrine, and with in planta accumulation of phenylalkylamines. Co-expression of recombinant genes encoding PaNMT and an ω-transaminase (PP2799) from Pseudomonas putida in Escherichia coli enabled the conversion of exogenous (R)-phenylacetylcarbinol (PAC) and (S)-PAC to ephedrine and pseudoephedrine, respectively. Our work further demonstrates the utility of plant biochemical genomics for the isolation of key enzymes that facilitate microbial engineering for the production of medicinally important metabolites.

Heterodimeric O-methyltransferases involved in the biosynthesis of noscapine in opium poppy.

Noscapine biosynthesis in opium poppy involves three characterized O-methyltransferases (OMTs) and a fourth responsible for the 4'-methoxyl on the phthalide isoquinoline scaffold. The first three enzymes are homodimers, whereas the latter is a heterodimer encoded by two linked genes (OMT2 and OMT3). Neither OMT2 nor OMT3 form stable homodimers, but yield a substrate-specific heterodimer when their genes are co-expressed in Escherichia coli. The only substrate, 4'-O-desmethyl-3-O-acetylpapaveroxine, is a seco-berbine pathway intermediate that undergoes ester hydrolysis subsequent to 4'-O-methylation leading to the formation of narcotine hemiacetal. In the absence of 4'-O-methylation, a parallel pathway yields narcotoline hemiacetal. Dehydrogenation produces noscapine and narcotoline from the corresponding hemiacetals. Phthalide isoquinoline intermediates with a 4'-hydroxyl (i.e. narcotoline and narcotoline hemiacetal), or the corresponding 1-hydroxyl on protoberberine intermediates, were not accepted. Norcoclaurine 6OMT, which shares 81% amino acid sequence identity with OMT3, also formed a functionally similar heterodimer with OMT2. Suppression of OMT2 transcript levels in opium poppy increased narcotoline accumulation, whereas reduced OMT3 transcript abundance caused no detectable change in the alkaloid phenotype. Opium poppy chemotype Marianne accumulates high levels of narcotoline and showed no detectable OMT2:OMT3 activity. Compared with the active subunit from the Bea's Choice chemotype, Marianne OMT2 exhibited a single S122Y mutation in the dimerization domain that precluded heterodimer formation based on homology models. Both subunits contributed to the formation of the substrate-binding domain, although site-directed mutagenesis revealed OMT2 as the active subunit. The occurrence of physiologically relevant OMT heterodimers increases the catalytic diversity of enzymes derived from a smaller number of gene products.

Codeinone reductase isoforms with differential stability, efficiency and product selectivity in opium poppy.

Codeinone reductase (COR) catalyzes the reversible NADPH-dependent reduction of codeinone to codeine as the penultimate step of morphine biosynthesis in opium poppy (Papaver somniferum). It also irreversibly reduces neopinone, which forms by spontaneous isomerization in aqueous solution from codeinone, to neopine. In a parallel pathway involving 3-O-demethylated analogs, COR converts morphinone to morphine, and neomorphinone to neomorphine. Similar to neopine, the formation of neomorphine by COR is irreversible. Neopine is a minor substrate for codeine O-demethylase (CODM), yielding morphine. In the plant, neopine levels are low and neomorphine has not been detected. Silencing of CODM leads to accumulation of upstream metabolites, such as codeine and thebaine, but does not result in a shift towards higher relative concentrations of neopine, suggesting a mechanism in the plant for limiting neopine production. In yeast (Saccharomyces cerevisiae) engineered to produce opiate alkaloids, the catalytic properties of COR lead to accumulation of neopine and neomorphine as major products. An isoform (COR-B) was isolated from opium poppy chemotype Bea's Choice that showed higher catalytic activity than previously characterized CORs, and it yielded mostly neopine in vitro and in engineered yeast. Five catalytically distinct COR isoforms (COR1.1-1.4 and COR-B) were used to determine sequence-function relationships that influence product selectivity. Biochemical characterization and site-directed mutagenesis of native COR isoforms identified four residues (V25, K41, F129 and W279) that affected protein stability, reaction velocity, and product selectivity and output. Improvement of COR performance coupled with an ability to guide pathway flux is necessary to facilitate commercial production of opiate alkaloids in engineered microorganisms.

Production of methylparaben in Escherichia coli.

Since the 1930s, parabens have been employed widely as preservatives in food, pharmaceutical, and personal care products. These alkyl esters of benzoic acid occur naturally in a broad range of plant species, where they are thought to enhance overall fitness through disease resistance and allelopathy. Current manufacture of parabens relies on chemical synthesis and the processing of 4-hydroxybenzoate as a precursor. A variety of bio-based production platforms have targeted 4-hydroxybenzoate for a greener alternative to chemical manufacturing, but parabens have yet to be made in microbes. Here, we deploy the plant enzyme benzoic acid carboxyl methyltransferase together with four additional recombinant enzymes to produce methylparaben in Escherichia coli. The feasibility of a tyrosine-dependent route to methylparaben is explored, establishing a framework for linking paraben production to emerging high-tyrosine E. coli strains. However, our use of a unique plant enzyme for bio-based methylparaben biosynthesis is potentially applicable to any microbial system engineered for the manufacture of 4-hydroxybenzoate.

Benzylisoquinoline Alkaloids Biosynthesis in Sacred Lotus.

Sacred lotus (Nelumbo nucifera Gaertn.) is an ancient aquatic plant used throughout Asia for its nutritional and medicinal properties. Benzylisoquinoline alkaloids (BIAs), mostly within the aporphine and bisbenzylisoquinoline structural categories, are among the main bioactive constituents in the plant. The alkaloids of sacred lotus exhibit promising anti-cancer, anti-arrhythmic, anti-HIV, and anti-malarial properties. Despite their pharmacological significance, BIA metabolism in this non-model plant has not been extensively investigated. In this review, we examine the diversity of BIAs in sacred lotus, with an emphasis on the distinctive stereochemistry of alkaloids found in this species. Additionally, we discuss our current understanding of the biosynthetic genes and enzymes involved in the formation of 1-benzylisoquinoline, aporphine, and bisbenzylisoquinoline alkaloids in the plant. We conclude that a comprehensive functional characterization of alkaloid biosynthetic enzymes using both in vitro and in vivo methods is required to advance our limited knowledge of BIA metabolism in the sacred lotus.

Isolation and Characterization of Reticuline N-Methyltransferase Involved in Biosynthesis of the Aporphine Alkaloid Magnoflorine in Opium Poppy.

Benzylisoquinoline alkaloids are a large group of plant-specialized metabolites displaying an array of biological and pharmacological properties associated with numerous structural scaffolds and diverse functional group modification. N-Methylation is one of the most common tailoring reactions, yielding tertiary and quaternary pathway intermediates and products. Two N-methyltransferases accepting (i) early 1-benzylisoquinoline intermediates possessing a secondary amine and leading to the key branch-point intermediate (S)-reticuline and (ii) downstream protoberberines containing a tertiary amine and forming quaternary intermediates destined for phthalideisoquinolines and antimicrobial benzo[c]phenanthridines were previously characterized. We report the isolation and characterization of a phylogenetically related yet functionally distinct N-methyltransferase (NMT) from opium poppy (Papaver somniferum) that primarily accepts 1-benzylisoquinoline and aporphine substrates possessing a tertiary amine. The preferred substrates were the R and S conformers of reticuline and the aporphine (S)-corytuberine, which are proposed intermediates in the biosynthesis of magnoflorine, a quaternary aporphine alkaloid common in plants. Suppression of the gene encoding reticuline N-methyltransferase (RNMT) using virus-induced gene silencing in opium poppy resulted in a significant decrease in magnoflorine accumulation and a concomitant increase in corytuberine levels in roots. RNMT transcript levels were also most abundant in roots, in contrast to the distribution of transcripts encoding other NMTs, which occur predominantly in aerial plant organs. The characterization of a third functionally unique NMT involved in benzylisoquinoline alkaloid metabolism will facilitate the establishment of structure-function relationships among a large group of related enzymes.

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

Benzylisoquinoline alkaloids (BIAs) are produced in a wide variety of plants and include many common analgesic, antitussive, and anticancer compounds. Several members of a distinct family of S-adenosylmethionine (SAM)-dependent N-methyltransferases (NMTs) play critical roles in BIA biosynthesis, but the molecular basis of substrate recognition and catalysis is not known for NMTs involved in BIA metabolism. To address this issue, the crystal structure of pavine NMT from Thalictrum flavum was solved using selenomethionine-substituted protein (dmin = 2.8 Å). Additional structures were determined for the native protein (dmin = 2.0 Å) as well as binary complexes with SAM (dmin = 2.3 Å) or the reaction product S-adenosylhomocysteine (dmin = 1.6 Å). The structure of a complex with S-adenosylhomocysteine and two molecules of tetrahydropapaverine (THP; one as the S conformer and a second in the R configuration) (dmin = 1.8 Å) revealed key features of substrate recognition. Pavine NMT converted racemic THP to laudanosine, but the enzyme showed a preference for (±)-pavine and (S)-reticuline as substrates. These structures suggest the involvement of highly conserved residues at the active site. Mutagenesis of three residues near the methyl group of SAM and the nitrogen atom of the alkaloid acceptor decreased enzyme activity without disrupting the structure of the protein. The binding site for THP provides a framework for understanding substrate specificity among numerous NMTs involved in the biosynthesis of BIAs and other specialized metabolites. This information will facilitate metabolic engineering efforts aimed at producing medicinally important compounds in heterologous systems, such as yeast.

Tying the knot: occurrence and possible significance of gene fusions in plant metabolism and beyond.

Gene fusions have recently attracted attention especially in the field of plant specialized metabolism. The occurrence of a gene fusion, in which originally separate gene products are combined into a single polypeptide, often corresponds to the functional association of individual components within a single metabolic pathway. Examples include gene fusions implicated in benzylisoquinoline alkaloid (BIA), terpenoid, and amino acid biosynthetic pathways, in which distinct domains within a fusion catalyze consecutive, yet independent reactions. Both genomic and transcriptional mechanisms result in the fusion of gene products, which can include partial or complete domain repeats and extensive domain shuffling as evident in the BIA biosynthetic enzyme norcoclaurine synthase. Artificial gene fusions are commonly deployed in attempts to engineer new or improved pathways in plants or microorganisms, based on the premise that fusions are advantageous. However, a survey of functionally characterized fusions in microbial systems shows that the functional impact of fused gene products is not straightforward. For example, whereas enzyme fusions might facilitate the metabolic channeling of unstable intermediates, this channeling can also occur between tightly associated independent enzymes. The frequent occurrence of both fused and unfused enzymes in plant and microbial metabolism adds additional complexity, in terms of both pathway functionality and evolution.

Acetylation serves as a protective group in noscapine biosynthesis in opium poppy.

We have characterized four sequential enzymes that transform 1-hydroxy-N-methylcanadine to narcotoline hemiacetal, completing our elucidation of noscapine biosynthesis in opium poppy. Two cytochromes P450 catalyze hydroxylations at C13 and C8 on the protoberberine scaffold, the latter step inducing ring opening and the formation of an aldehyde moiety. Acetylation at C13 before C8 hydroxylation introduces a protective group subsequently hydrolyzed by a carboxylesterase, which triggers rearrangement to a cyclic hemiacetal.

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

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.