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

• Published in: The Journal of biological chemistry 2018-08, Vol.293 (35), p.13364-13376
• Main Authors: Morris, Jeremy S., Groves, Ryan A., Hagel, Jillian M., Facchini, Peter J.
• Format: Article
• Language: English
• Subjects: biochemical genomics; Biosynthetic Pathways; Ephedra sinica; Ephedra sinica - enzymology; Ephedra sinica - genetics; Ephedra sinica - metabolism; Ephedrine - metabolism; genomics; Methyltransferases - genetics; Methyltransferases - metabolism; N-methyltransferase; natural product; plant biochemistry; Plant Biology; plant molecular biology; Pseudoephedrine - metabolism; Secondary Metabolism; secondary metabolite; substituted phenylalkylamine; Substrate Specificity; synthetic biology; Transcriptome
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.RA118.004067

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.