An (R)-specific N-methyltransferase involved in human morphine biosynthesis

Area of Science:

• Biochemistry
• Pharmacology
• Genetics

Background:

• Morphine biosynthesis, a complex alkaloid pathway, occurs in plants and animals.
• Benzylisoquinoline alkaloids are key biosynthetic precursors of morphine.
• Methyltransferases play a critical role in N-methylation during alkaloid biosynthesis.

Purpose of the Study:

• To identify human methyltransferases involved in the N-methylation of benzylisoquinoline alkaloids.
• To characterize the substrate specificity and kinetic parameters of identified human enzymes.
• To investigate the potential role of human enzymes in mammalian morphine biosynthesis.

Main Methods:

• Bioinformatic search for methyltransferases in the human genome.
• Purification of recombinant human PNMT (EC 2.1.1.28) and NMT (EC 2.1.1.49) using immobilized metal affinity chromatography.
• Enzymatic assays to determine catalytic activity and kinetic parameters using various benzylisoquinoline alkaloids and LC-MS/MS detection.
• In vivo experiments involving the injection of (R)-THP into mice and subsequent isolation of salutaridine from urine.

Main Results:

• Human PNMT showed no activity towards benzylisoquinoline alkaloids but was active with phenylethanolamine.
• Human NMT exhibited broad substrate acceptance for four tested benzylisoquinolines, with a strong preference for (R)-configured precursors.
• (R)-norreticuline was identified as the optimal substrate for human NMT, exhibiting higher catalytic activity than tryptamine.
• Isolation of salutaridine from mouse urine after (R)-THP injection provided in vivo evidence for mammalian morphine precursor biosynthesis.

Conclusions:

• Human NMT is an (R)-specific enzyme capable of N-methylating morphine precursors.
• The findings suggest that human NMT plays a role in mammalian morphine biosynthesis, potentially differing stereochemically and sequentially from plant pathways.
• This study provides evidence for the involvement of a specific human N-methyltransferase in the initial steps of morphine precursor formation in mammals.