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.

Figure 1.

Pathway leading to the formation of (S)-norcoclaurine, the central intermediate in the biosynthesis of BIAs in plants, from two molecules of l-Tyr. Dopamine is derived via decarboxylation and 3-hydroxylation of l-Tyr, although the prevailing reaction order is not known. Tyr/DOPA decarboxylase (TYDC) has been shown to accept l-Tyr and l-DOPA as substrates. However, the enzyme (1) responsible for the 3- hydroxylation of l-Tyr or tyramine has not been identified. 4-HPAA is suggested to result from the transamination of l-Tyr and the subsequent decarboxylation of 4-HPP. Tyr transaminase (2) and 4-HPP decarboxylase (3) activities have been reported in BIA-producing plants, but the corresponding enzymes have not been isolated. The condensation of dopamine and 4-HPPA is catalyzed by NCS.

Figure 2.

Unrooted neighbor-joining tree showing the phylogenetic relationships between opium poppy TyrAT and related plant proteins. Numbers in the tree refer to the bootstrap values for each node over 1,000 iterations. Numbers in parentheses show the percentage amino acid identity of each protein compared with TyrAT from opium poppy. The annotations and GenBank accession numbers of each protein are as follows: PsTyrAT, opium poppy TyrAT (GU370929); OsTyrAT, O. sativa japonica group putative nicotianamine aminotransferase (BAF95202); SpTyrAT, S. pennellii putative TyrAT (ADZ24702); RcTyrAT, R. communis putative TyrAT (XP_002517869); PtTyrAT, P. trichocarpa aminotransferase family protein (XP_002328046); SsTyrAT, S. scutellaridoides putative TyrAT (CAD30341); SmTyrAT, S. miltiorrhiza putative TyrAT (ABC60050); MtTyrAT, M. truncatula putative TyrAT (AAY85183); GmTyrAT, G. max putative TyrAT (AAY21813); CmTyrAT, melon aromatic amino acid transaminase (ADC45389); AtTyrAT-1, Arabidopsis coronatine-regulated TyrAT (TAT1; AAN15626); AtTyrAT-2, Arabidopsis Tyr:2-oxoglutarate aminotransferase (TAT3; NP_180058); AtTyrAT-3, Arabidopsis rooty/superroot1 protein (AAG37062); AtTyrAT-4, Arabidopsis TyrAT (NM_124776).

Figure 3.

Purification of His₆-tagged, recombinant PsTyrAT from E. coli total soluble protein extracts by cobalt-affinity chromatography. Elutions were performed using increasing imidazole concentrations: 10 mm (lane 1), 30 mm (lane 2), 50 mm (lane 3), and 100 mm (lane 4). Lanes contained 20 μL of each fraction, which were separated by SDS-PAGE and visualized using Commassie Brilliant Blue G-250 stain.

Figure 4.

Extracted ion chromatograms at m/z 179.1 for enzyme assays using native and heat-inactivated PsTyrAT protein, and the corresponding CID spectrum for the compound eluting at the retention time of 0.54 min. Enzyme assays contained 2 μg of purified, recombinant PsTyrAT protein incubated with 0.1 mm PLP, 0.1 mm EDTA, 0.3 mm α-ketoglutarate, and 3 mm l-Tyr for 1 h at 30°C. Boiled PsTyrAT protein was used as the heat-inactivated control. Using the native enzyme, MRM in negative mode showed peaks corresponding to 4-HPP using the fragment ions at m/z 151.1 and 107.1 for the precursor ion at m/z 179.1. No peaks corresponding to 4-HPP were detected using the heat-inactivated enzyme. CID in the range of m/z 20 to 200 confirmed that the extracted ion spectra were derived from 4-HPP by the presence of a fragment ion at m/z 107.1 (inset).

Figure 5.

Effect of pH on PsTyrAT activity. Assays were performed for 1 h at 30°C in HEPES buffer at the pH values indicated in the presence of 2 μg of purified enzyme, 3 mm l-Tyr, 0.1 mm PLP, 0.1 mm EDTA, and 0.5 mm α-ketoglutarate.

Figure 6.

Relative transcript abundance of PsTyrAT in different opium poppy organs. First-strand cDNAs were synthesized from total RNA and used as a template for RT-qPCR analysis. The transcript abundance of ubiquitin from opium poppy was used as an internal control, and relative values were normalized to the PsTyrAT transcript level in roots. Values represent means ± sd of triplicate experiments.

Figure 7.

Effect of reducing PsTyrAT transcript levels by VIGS in opium poppy plants. A, A 492-bp fragment of the PsTyrAT coding region was inserted into pTRV2 vector. Two-week-old poppy seedlings were coinfiltrated with pTRV1 and pTRV2-EV or with pTRV1 and pTRV2-TyrAT. After approximately 10 weeks, stem and latex samples were used to determine relative PsTyrAT transcript abundance and BIA levels, respectively. B, Ethidium bromide-stained agarose gel showing the detection of TRV2 coat protein transcripts in cDNAs synthesized from total stem RNA. C, Mean PsTyrAT transcript levels in plants infiltrated with pTRV2-TyrAT (black bar) and pTRV2-EV (white bar). D, Relative accumulation of total major BIAs in latex extracted from plants infiltrated with pTRV2-TyrAT (black bar) and pTRV2-EV (white bar). E, Relative accumulation of individual BIAs in latex extracted from plants infiltrated with pTRV-TyrAT (black bars) and pTRV2-EV (white bars). Values represent means ± sd of three technical replicates performed on each of three biological replicates for each of eight infiltrated plants.