Tyrosine Aminotransferase Contributes to Benzylisoquinoline Alkaloid Biosynthesis in Opium Poppy  

Abstract

Tyrosine aminotransferase (TyrAT) catalyzes the transamination of L-Tyr and a-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 a-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.

A Root-Expressed l-Phenylalanine:4-Hydroxyphenylpyruvate Aminotransferase Is Required for Tropane Alkaloid Biosynthesis in Atropa belladonna

“…Asterisks denote significant differences (**P < 0.01) as determined by Student's t test. the kinetic properties of ArATs previously determined to exhibit maximal activity with 2-oxoglutarate as the cosubstrate to assess their activities with aromatic keto-acids (Prabhu and Hudson, 2010;Lee and Facchini, 2011;Riewe et al, 2012).…”

“…The reported activity of several ArATs indicates they can use L-Tyr, L-Phe, and L-Trp to varying degrees as amino donors, with 2-oxoglutarate generally being the preferential amino acceptor (Prabhu and Hudson, 2010;Lee and Facchini, 2011;Riewe et al, 2012). More recently, Ph-PPY-AT and Cm-ArAT1 were shown to preferentially catalyze the formation of L-Phe using L-Tyr as the amino donor and phenylpyruvate as the amino acceptor (Yoo et al, 2013).…”

“…Full-length transcripts were obtained for Ab-ArAT1 to 5, which encode proteins of between 419 and 441 amino acids (46 to 48 kD) that share between 50 and 72% amino acid identity. The majority of putative plant ArATs remain uncharacterized but the closely related Arabidopsis proteins encoded by At5g36160 and At5g53970, Ps-TyrAT from Papaver somniferum, Ph-PPY-AT from petunia, and Cm-ArAT1 from melon (Cucumis melo) all utilize L-Phe and L-Tyr as amino donors (Prabhu and Hudson, 2010;Lee and Facchini, 2011;Riewe et al, 2012;Yoo et al, 2013). An amino acid alignment of the predicted protein sequence of the Ab-ArAT proteins and selected homologs indicates that each contains a conserved catalytic lysine that corresponds to residue 270 in Ab-ArAT1, together with conserved residues required for the binding of the pyridoxal-59-phosphate cofactor (Supplemental Figure 3).…”

Characterization of Three O-Methyltransferases Involved in Noscapine Biosynthesis in Opium Poppy

“…Total RNA from different organs of the opium poppy cv Bea's Choice was extracted, and cDNA was synthesized as described previously (Lee and Facchini, 2011). The relative abundance of candidate SOMT transcripts in each organ was determined by RT-qPCR analysis using the primers described in Supplemental Table S4.…”

An alternative pathway contributes to phenylalanine biosynthesis in plants via a cytosolic tyrosine:phenylpyruvate aminotransferase

“…Nevertheless, overexpression of a bacterial bifunctional chorismate mutase/prephenate dehydratase (PheA) in Arabidopsis resulted in significantly increased phenylalanine production 19 , implicating the presence of an aminotransferase capable of converting phenylpyruvate to phenylalanine in plants, as it occurs in the final step of the microbial phenylpyruvate pathway. Although several plant aminotransferases, mainly participating in aromatic amino-acid catabolism, have recently been described [20][21][22][23] , none of them have been demonstrated to function in phenylalanine biosynthesis.…”