Abstract
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, β-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.
Highlights
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
Combined tBLASTn and HMMer searches of an E. sinica stem transcriptome [11] using six canonical plant NMT proteins as queries yielded 27 candidate enzymes potentially able to catalyze the N-methylation of phenylalkylamine intermediates in the biosynthesis ofephedrine (Table S1; Fig. S1)
Full-length open reading frames corresponding to 18 genes encoding candidate NMT enzymes were amplified by RT-PCR using total RNA from E. sinica stems (Table S1), 16 of which yielded sufficient His6-tagged recombinant protein in E. coli to allow for partial purification using a cobalt–affinity resin (Fig. S2)
Summary
Facchini From the Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada 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. 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. Radiolabeled precursor feeding experiments, the occurrence (or absence) of purported intermediates, the detection of key enzyme activities and other lines of plant-based evidence, Ephedra alkaloid biosynthesis is thought to involve multiple steps, including stereoselective reduction to either (1S,2S)- or (1R,2S)-form isomers, followed by N-methylation to yield pseudoephedrine and ephedrine, respectively (Fig. 1) [5, 10, 11]. Our discovery completes the set of enzymes required for the potential commercial production of (pseudo)ephedrine, and other phenylalkylamines, in engineered microorganisms
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