title: Tyrosine aminotransferase contributes to benzylisoquinoline alkaloid biosynthesis in opium poppy.
abstract: Tyrosine aminotransferase (TyrAT) catalyzes the transamination of L-Tyr and α-ketoglutarate, yielding 4-hydroxyphenylpyruvic acid and L-glutamate. The decarboxylation product of 4-hydroxyphenylpyruvic acid, 4-hydroxyphenylacetaldehyde, is a precursor to a large and diverse group of natural products known collectively as benzylisoquinoline alkaloids (BIAs). We have isolated and characterized a TyrAT cDNA from opium poppy (Papaver somniferum), which remains the only commercial source for several pharmaceutical BIAs, including codeine, morphine, and noscapine. TyrAT belongs to group I pyridoxal 5'-phosphate (PLP)-dependent enzymes wherein Schiff base formation occurs between PLP and a specific Lys residue. The amino acid sequence of TyrAT showed considerable homology to other putative plant TyrATs, although few of these have been functionally characterized. Purified, recombinant TyrAT displayed a molecular mass of approximately 46 kD and a substrate preference for L-Tyr and α-ketoglutarate, with apparent K(m) values of 1.82 and 0.35 mm, respectively. No specific requirement for PLP was detected in vitro. Liquid chromatography-tandem mass spectrometry confirmed the conversion of L-Tyr to 4-hydroxyphenylpyruvate. TyrAT gene transcripts were most abundant in roots and stems of mature opium poppy plants. Virus-induced gene silencing was used to evaluate the contribution of TyrAT to BIA metabolism in opium poppy. TyrAT transcript levels were reduced by at least 80% in silenced plants compared with controls and showed a moderate reduction in total alkaloid content. The modest correlation between transcript levels and BIA accumulation in opium poppy supports a role for TyrAT in the generation of alkaloid precursors, but it also suggests the occurrence of other sources for 4-hydroxyphenylacetaldehyde.
authors.affiliation: Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
authors.forename: Eun-Jeong
authors.initials: EJ
authors.lastname: Lee
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Facchini
mesh_terms: D044182:Benzylisoquinolines / Q000737:chemistry / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009892:Opium / Q000378:metabolism*; D010207:Papaver / Q000201:enzymology* / Q000235:genetics; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification; D014444:Tyrosine Transaminase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D044182:Benzylisoquinolines; D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D009892:Opium; D014444:Tyrosine Transaminase