A root-expressed L-phenylalanine:4-hydroxyphenylpyruvate aminotransferase is required for tropane alkaloid biosynthesis in Atropa belladonna

Abstract

The tropane alkaloids, hyoscyamine and scopolamine, are medicinal compounds that are the active components of several therapeutics. Hyoscyamine and scopolamine are synthesized in the roots of specific genera of the Solanaceae in a multistep pathway that is only partially elucidated. To facilitate greater understanding of tropane alkaloid biosynthesis, a de novo transcriptome assembly was developed for Deadly Nightshade (Atropa belladonna). Littorine is a key intermediate in hyoscyamine and scopolamine biosynthesis that is produced by the condensation of tropine and phenyllactic acid. Phenyllactic acid is derived from phenylalanine via its transamination to phenylpyruvate, and mining of the transcriptome identified a phylogenetically distinct aromatic amino acid aminotransferase (ArAT), designated Ab-ArAT4, that is coexpressed with known tropane alkaloid biosynthesis genes in the roots of A. belladonna. Silencing of Ab-ArAT4 disrupted synthesis of hyoscyamine and scopolamine through reduction of phenyllactic acid levels. Recombinant Ab-ArAT4 preferentially catalyzes the first step in phenyllactic acid synthesis, the transamination of phenylalanine to phenylpyruvate. However, rather than utilizing the typical keto-acid cosubstrates, 2-oxoglutarate, pyruvate, and oxaloacetate, Ab-ArAT4 possesses strong substrate preference and highest activity with the aromatic keto-acid, 4-hydroxyphenylpyruvate. Thus, Ab-ArAT4 operates at the interface between primary and specialized metabolism, contributing to both tropane alkaloid biosynthesis and the direct conversion of phenylalanine to tyrosine.

Figure 1.

The Proposed Tropane Alkaloid Biosynthetic Pathway in A. belladonna and Related Solanaceous Species. Enzyme name abbreviations are shown. ODC, ornithine decarboxylase; PMT, putrescine methyl transferase; MPO, methyl putrescine oxidase; TR I, tropinone reductase I; TR II, tropinone reductase II; CYP80F1, littorine mutase; H6H, hyoscyamine-6-hydroxylase. Unknown steps between the N-methyl-Δ¹-pyrrolinium cation and tropinone are shown as a dashed arrow. Ab-ArAT4, the subject of this study, is shown in bold.

Figure 2.

Functional Annotation of A. belladonna Transcripts. A. belladonna unigenes were aligned with annotated A. belladonna sequences in GenBank, UniRef, the predicted Arabidopsis proteome, and the Pfam domain database. For the UniRef, Arabidopsis, and Pfam results, the percentage of total A. belladonna unigenes with a match in these three databases is reported. For the A. belladonna GenBank results, the percentage of GenBank entries with a match to the A. belladonna unigene set is reported.

Figure 3.

Cluster of Orthologous and Paralogous Gene Families in Four Plant Species Identified by OrthoMCL. Number of clusters (c) and genes (g) for each orthologous group are presented. Original data are provided in Supplemental Data Set 1.

Figure 4.

Phylogenetic Relationship of A. belladonna ArATs and Related Proteins. A Maximum Likelihood phylogenetic tree was constructed as described in Methods. Shading corresponds to putative orthologous proteins of the Solanaceae family defined as clades A through E. The identities of the proteins are provided in Supplemental Table 1, and the multiple sequence alignment used to construct the phylogeny is provided as Supplemental Data Set 2. Bootstrap values derived from 2000 replicates are shown on the nodes of the tree. Ab-ArAT4 is highlighted in bold.

Figure 5.

Silencing of ArAT4 Affects Tropane Alkaloid Biosynthesis. (A) Abundance of the tropane alkaloids hyoscyamine and scopolamine, together with selected intermediates and precursors in TRV2 empty vector control lines and ArAT4 VIGS lines. Data are presented as the mean n = 17 or 21 ± se. Asterisks denote significant differences (***P < 0.001) as determined by Student’s t test. (B) Relative expression level of ArATs in TRV2 empty vector control lines and ArAT4 VIGS lines. Data were obtained by quantitative RT-PCR and are presented as the mean of six biological and three technical replicates with the expression level of ArAT4 in TRV2 empty vector control lines set to 1 ± se. The six plants selected for gene expression analysis represent individuals with phenyllactic acid levels closest to the median values for the given genotypes presented in (A). Asterisks denote significant differences (*P < 0.05; **P < 0.01) as determined by Student’s t test. [See online article for color version of this figure.]

Figure 6.

l-Phenylalanine:4-Hydroxyphenylpyruvate Aminotransferase Activity in ArAT4-Silenced Lines. (A) Summary of the preferred reaction of ArAT4. (B) ArAT activity was determined through monitoring the production of l-Tyr using l-Phe and 4-HPP as substrates in reaction mixes containing crude protein extracted from TRV2 and TRV2:ArAT4 VIGS lines. Data are presented as the mean of three technical replicates derived from a pooled set of three biological replicates for each genotype, ± se. Asterisks denote significant differences (**P < 0.01) as determined by Student’s t test.

Figure 7.

Ab-ArAT4 Acts at the Interface of Tropane Alkaloid Biosynthesis and Aromatic Amino Acid Metabolism. Ab-ArAT4 participates in the catabolism of l-Phe to direct phenylpyruvate (PPY) and phenyllactate toward tropane alkaloid biosynthesis and simultaneously catalyzes the anabolism of l-Tyr. The characterized enzymes of aromatic amino acid metabolism are shown in bold. Ab-ArAT4, A. belladonna Aromatic Aminotransferase 4; CM, chorismate mutase, ADH; arogenate dehydrogenase; ADT, arogenate dehydratase; PPA-AT, prephenate aminotransferase. [See online article for color version of this figure.]