Letter Cite This: Org. Lett. 2018, 20, 7807−7810
pubs.acs.org/OrgLett
A Phenylpyruvic Acid Reductase Is Required for Biosynthesis of Tropane Alkaloids Fei Qiu,† Chunxian Yang,† Lina Yuan,† Dan Xiang,† Xiaozhong Lan,‡ Min Chen,§ and Zhihua Liao*,† †
Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), School of Life Sciences, Southwest University, Chongqing 400715, China ‡ TAAHC-SWU Medicinal Plant Joint R&D Centre, Xizang Agricultural and Husbandry College, Nyingchi of Tibet 860000, China § College of Pharmaceutical Sciences, Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Ministry of Education), Southwest University, Chongqing 400715, China
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S Supporting Information *
ABSTRACT: Solanaceous medicinal plants produce tropane alkaloids (TAs). We discovered a novel gene from Atropa belladonna, AbPPAR, which encodes a phenylpyruvic acid reductase required for TA biosynthesis. AbPPAR was specifically expressed in root pericycles and endodermis. AbPPAR was shown to catalyze reduction of phenylpyruvic acid to phenyllactic acid, a precursor of TAs. Suppression of AbPPAR disrupted TA biosynthesis through reduction of phenyllactic acid levels. In summary, we identified a novel enzyme involved in TA biosynthesis.
T
Scheme 1. Proposed Biosynthetic Pathway of Tropane Alkaloids in Solanaceaea
he tropane alkaloids (TAs) of Solanaceae, such as hyoscyamine (1), anisodamine (2), and scopolamine (3), are widely used as anticholinergic agents to treat motion sickness, postoperative nausea, arrhythmia, and Parkinson’s diseases.1,2 These TAs are specifically produced in genera of Solanaceae including Atropa, Datura, Hyoscyamus, and etc.3−5 The medicinal plants of Solanaceae are the only commercial source for producing these valuable compounds, but the alkaloid contents in planta are very low, thus prompting plant scientists to find new approaches to produce TAs. Metabolic engineering and synthetic biology offer promising approaches to enhance TA production. On a more fundamental level, however, elucidation of the entire TA biosynthetic pathway is essential. Although TA biogenic routes are clear (Scheme 1), TA biosynthesis is not completely understood. Fortunately, several TA biosynthesis enzymes have been identified from different TA-producing plant species of Solanaceae (Scheme 1). Such enzymes include putrescine N-methyltransferase (PMT), Nmethylputrescine oxidase (MPO), tropinone reductase I (TRI), littorine mutase (LM, CYP80F1), aromatic amino acid aminotransferase (AT4), and hyoscyamine 6β-hydroxylase (H6H). PMT catalyzes N-methylation of putrescine to produce N-methylputrescine, which is subsequently converted to 4-methylaminobutanal by MPO. After unknown enzymatic reactions, tropinone is synthesized. TRI converts tropinone to tropine, providing the tropane moiety of TAs. Further, tropine is condensed with a phenyllactic acid moiety to produce littorine (4). AT4 catalyzes the transamination of phenylalanine to produce phenylpyruvic acid (5) that is then reduced © 2018 American Chemical Society
a AR, arginase. ODC, ornithine decarboxylase. PMT, putrescine Nmethyltransferase. MPO, N-methylputrescine oxidase. TRI, tropineforming reductase. CYP80F1, littorine mutase. H6H, hyoscyamine 6β-hydroxylase. AT4, aromatic amino acid aminotransferase. PPAR, phenylpyruvic acid reductase.
to phenyllactic acid (6). Littorine mutase CYP80F1 converts 4 into hyoscyamine aldehyde, which is the direct precursor of 1. Received: October 11, 2018 Published: December 4, 2018 7807
DOI: 10.1021/acs.orglett.8b03236 Org. Lett. 2018, 20, 7807−7810
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Organic Letters
thesis genes were specifically or highly expressed in secondary roots, and AbPPAR was exclusively expressed in roots, with much higher levels in the secondary roots (Figure 1A).
Finally, H6H catalyzes 6β-hydroxylation of 1 to 2, as well as the subsequent epoxidation of 2 to 3.4,6−11 To date, the full TA biosynthetic pathway has not been completely resolved. Uncharacterized enzymes involved in TA biosynthesis may catalyze the formation of tropinone, the reduction of 5 to 6, the production of 4, and the conversion of hyoscyamine aldehyde into 1. Isotope-labeling experiments clearly demonstrated 6 was incorporated at high levels into TAs including 4, 1, and 3, confirming that 6 is a precursor of TAs.12 However, the enzyme catalyzing the reduction of 5 has not been found in the plant kingdom. In this study, we identified a novel root-expressed gene encoding a phenylpyruvic acid reductase from Atropa belladonna (AbPPAR), which is required for TA biosynthesis. It is well known that hydroxyphenylpyruvate reductase (HPPR) catalyzes the reduction of hydroxyphenylpyruvate to its respective lactate.13 However, HPPRs have not been shown to reduce 5 to form 6 in plants. To date, a phenylpyruvic acid reductase has not been found in plants. Theoretically, the reduction of 5 to 6 is similar to the reaction catalyzed by HPPR. In other words, phenylpyruvic acid reductase might be a homologue of HPPRs. Biochemically, HPPR belongs to the formate/glycerate dehydrogenase like family of proteins that contain a hydroxyl acid dehydrogenase (HAD) domain (PF00389). We used PF00389 in a Hidden Markov Model (HMM) program to search sequenced transcriptome data of A. belladonna and identified 38 sequences belonging to 16 unigenes. TAs are specifically synthesized in secondary roots of A. belladonna, due to high or specific expression of TA biosynthesis genes in roots. Therefore, we predicted that unidentified TA biosynthesis genes might also be specifically or highly expressed in secondary roots. To this end, the expression relationship between previously identified TA biosynthesis genes and the 16 unigenes were analyzed by a Multiple Experiment Viewer. Solely, one of the 16 unigenes, aba_locus_14073_iso_2_len_1166_ver_2, was highly expressed in secondary roots and not expressed in stems and leaves (Supporting Information (SI) Figure S1), suggesting that it might be the candidate phenylpyruvic acid reductase gene (AbPPAR). To explore this possibility, full-length AbPPAR cDNA (MK121981) was isolated and used for functional identification (Figure S2). A phylogenetic analysis indicated that plant and microbial HADs were divided into different clades (Figure S3). Plant HPPR sequences were highly conserved (81.5% identity). In the clade of plant HADs, AbPPAR was clearly separated from the group of HPPRs, suggesting functional divergence between AbPPAR and plant HPPRs. Sequence comparison showed that AbPPAR had low similarity (
