Evolutionary recruitment of a flavin-dependent monooxygenase for the detoxification of host plant-acquired pyrrolizidine alkaloids in the alkaloid-defended arctiid moth Tyria jacobaeae Claudia Naumann, Thomas Hartmann, and Dietrich Ober* Institut fu¨r Pharmazeutische Biologie der Technischen Universita¨t Braunschweig, Mendelssohnstrasse 1, D-38106 Braunschweig, Germany Edited by May R. Berenbaum, University of Illinois at Urbana-Champaign, IL, and approved February 26, 2002 (received for review December 17, 2001) Larvae of Tyria jacobaeae feed solely upon the pyrrolizidine alkaloid- effects of PAs because of high levels of N-oxide forming FMO containing plant Senecio jacobaea. Ingested pyrrolizidine alkaloids activity and low levels of bioactivating P450 (9). (PAs), which are toxic to unspecialized insects and vertebrates, are Herbivorous insects also can cope with xenobiotics by means of efficiently N-oxidized in the hemolymph of T. jacobaeae by sene- P450s, as evidenced by resistance to commercial insecticides (15). cionine N-oxygenase (SNO), a flavin-dependent monooxygenase However, during evolutionary adaptation to their food plants, (FMO) with a high substrate specificity for PAs. Peptide microse- herbivorous insects have developed specific mechanisms to tolerate quences obtained from purified T. jacobaeae SNO were used to clone specific plant defense chemicals, often involving P450s. In one the corresponding cDNA, which was expressed in active form in example, Papilio polyxenes, the black swallowtail, feeds on Apiaceae Escherichia coli. T. jacobaeae SNO possesses a signal peptide charac- species containing phototoxic furanocoumarins, which are effi- teristic of extracellular proteins, and it belongs to a large family of ciently detoxified in the insect by a P450 monooxygenase. This mainly FMO-like sequences of mostly unknown function, including enzyme not only shows high substrate specificity for the furano- two predicted Drosophila melanogaster gene products. The data coumarins occurring in the host plant (16), but it also is specifically indicate that the gene for T. jacobaeae SNO, highly specific for toxic induced by these compounds (17) by means of the xanthotoxin- pyrrolizidine alkaloids, was recruited from a preexisting insect-spe- response element, which was identified in the promoter region of cific FMO gene family of hitherto unknown function. The enzyme this inducible P450 enzyme (18). In another example, Drosophila allows the larvae to feed on PA-containing plants and to accumulate mettleri, one of four Drosophila species endemic to the Sonoran predation-deterrent PAs in the hemolymph. desert in the southwestern United States, lives and breeds in soils soaked by rot exudate of the saguaro cactus, which contains toxic EVOLUTION N-oxidation ͉ alkaloid sequestering insects ͉ insect adaptation ͉ chemical isoquinoline alkaloids that accumulate in the soil because of water defense ͉ gene recruitment evaporation (19). D. mettleri’s unique isoquinoline alkaloid resis- tance involves a cytochrome P450 monooxygenase that is specifi- cally induced by isoquinolines from saguaro (20), but not from other arvae of the European cinnabar moth Tyria jacobaeae are cacti (20). Thus, P. polyxenes and D. mettleri each possess at least one Lspecialized to tansy ragwort (Senecio jacobaea L.) as food P450 enzyme whose substrate specificity and inducibility evolved in plant. Pyrrolizidine alkaloids (PAs) present in this plant are response to specific plant defense compounds (‘‘secondary metab- sequestered by the larvae and are retained during all stages of olites’’) present in the respective host plant. metamorphosis (1, 2). PAs are typical plant secondary com- Tyria jacobaeae larvae do not simply detoxify PAs by N-oxidation, pounds thought to play an important role as defensive chemicals but store the nontoxic N-oxides as predation deterrents (Fig. 1; 21). in the plant and its adapted insect herbivores (3). PAs are The larvae advertise their toxic PAs by a striking yellow-black hepatotoxic and carcinogenic to vertebrates and mutagenic or warning coloration. N-oxidation is only one of the different, re- genotoxic to insects and even to yeast (4); they also show strong cently identified strategies insects developed in evolution to safely antifeedant activity against various herbivores (5). In species of handle plant-acquired PAs for their own defense (22, 23). The the genus Senecio (Asteraceae), senecionine N-oxide is synthe- enzyme responsible for the N-oxidation of tertiary PAs in Tyria is sized in the roots as common PA-backbone structure, which senecionine N-oxygenase (SNO), which is a reduced nicotinamide- subsequently is transported to other organs and tissues where it adenine dinucleotide phosphate (NADPH)-dependent flavin is chemically modified into the species-specific PA patterns (6). monooxygenase (FMO), not a cytochrome P450 enzyme (21). In In the gut of mammalian herbivores feeding on PA-containing contrast to the intensively studied mammalian multisubstrate plants, the PA N-oxides are easily reduced, yielding the respec- FMOs (24), the Tyria enzyme is strictly substrate specific. It tive free bases, which are passively absorbed and thus become N-oxidizes only protoxic PAs, i.e., PAs with structural features accessible to the bioactivating microsomal cytochrome P450- dependent monooxygenases (P450) of liver. These enzymes are part of xenobiotic metabolism in vertebrates (7, 8) and transform This paper was submitted directly (Track II) to the PNAS office. the tertiary PAs to unstable pyrroles, which are highly reactive Abbreviations: PA, pyrrolizidine alkaloid; P450, cytochrome P450-dependent monooxy- alkylating agents. N-oxidation was shown to be a second pathway genase; NADPH, nicotinamide-adenine dinucleotide phosphate; FMO, flavin-dependent monooxygenase; SNO, senecionine N-oxygenase; RACE, rapid amplification of cDNA ends. of PA metabolism in mammals, catalyzed by flavin-dependent Data deposition: The sequence reported in this paper has been deposited in the EMBL monooxygenases (FMOs; ref. 9) or by an isoform of P450s (10, Nucleotide Sequence Database (accession no. AJ420233). 11). The potentially toxic tertiary PAs are oxidized to their *To whom reprint requests should be addressed. E-mail: [email protected]. N-oxides, which no longer serve as substrates for bioactivating The publication costs of this article were defrayed in part by page charge payment. This P450s (10, 12). In contrast to most mammals, guinea pigs, sheep, article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. and hamsters (9, 13, 14) exhibit a high resistance against the toxic §1734 solely to indicate this fact. www.pnas.org͞cgi͞doi͞10.1073͞pnas.082674499 PNAS ͉ April 30, 2002 ͉ vol. 99 ͉ no. 9 ͉ 6085–6090 Downloaded by guest on September 23, 2021 jacobaeae was recruited from a preexisting family of FMO-like proteins of hitherto unknown functions. Materials and Methods Radiochemicals. [N-Methyl-3H]atropine (87 Ci͞mmol) was pur- chased from DuPont. Pyrrolizidine alkaloids applied as enzyme substrates were obtained as described (21). Purification and Microsequencing of SNO. SNO was purified from hemolymph of T. jacobaeae as described (21). The purified enzyme was transferred to a poly(vinylidene difluoride) mem- brane (Millipore) and was subjected to microsequencing. Pep- tides of purified SNO obtained by digestion with trypsin were separated by means of HPLC on a Vydac C18 column (4.6 mm ϫ 150 mm) and sequenced according to ref. 26. RNA Isolation and cDNA Synthesis. Total RNA was isolated by using the RNeasy Mini Kit (Qiagen) from one-week-old larvae of T. jacobaeae and of the larvae and adults of D. melanogaster frozen in liquid nitrogen. Total RNA (2 ␮g) was reverse-transcribed by Superscript II (Invitrogen) in a total volume of 50 ␮l by using an ␮ oligo(dT)17 primer (0.1 M; Table 1). PCR with Degenerate Primers. Degenerate primers (P1–6) were constructed according to peptides resulting from partial sequencing Fig. 1. PA N-oxides ingested from plants are reduced in the gut. In their of the SNO protein (Table 1). Amplification of 1 ␮l of cDNA was tertiary form as lipophilic free bases, they permeate passively the membrane performed with Taq DNA Polymerase (Qiagen, Chatsworth, CA) and are trapped in the hemolymph by efficient re-N-oxidation catalyzed by in a total volume of 25 ␮l by using all possible combinations of SNO of T. jacobaeae (A). SNO is an NADPH-dependent monooxygenase (B). ␮ forward and reverse primers (2.0 M each), including oligo(dT)17 primer (1.0 ␮M). A touch-down protocol with decreasing annealing temperatures from 60°Cto45°C(0.5°C per cycle, 45°C constant for required by substrates of the bioactivating cytochrome P450 en- 20 further cycles) was used for the temperature program . With zymes, indicating that the major function of SNO is to maintain primers P5 and oligo(dT)17, a 554-bp fragment was amplified, which sequestered protoxic PAs as nontoxic N-oxides. No functionally subsequently was electrophoretically purified, subcloned by using characterized FMO is known from insects (15, 25). To trace the the TOPO TA cloning kit (Invitrogen) according to the manufac- evolutionary origin of SNO, we have obtained peptide sequences turer’s instructions, and sequenced. from the purified T. jacobaeae enzyme, cloned the cDNA, ex- pressed the active protein in E. coli and investigated its gene Amplification of the 5؅-cDNA End of SNO. For identification of the phylogeny. The data indicate that the PA-specific SNO of T.