The two Faces of Pyrrolizidine Alkaloids: the Role of the Tertiary Amine and its N‐Oxide in Chemical Defense of Insects with Acquired Plant Alkaloids

Abstract

Larvae of Creatonotos transiens (Lepidoptera, Arctiidae) and Zonocerus variegatus (Orthoptera, Pyrgomorphidae) ingest 14C-labeled senecionine and its N-oxide with the same efficiency but sequester the two tracers exclusively as N-oxide. Larvae of the non-sequestering Spodoptera littoralis eliminate efficiently the ingested alkaloids. During feeding on the two alkaloidal forms transient levels of senecionine (but not of the N-oxide) are built up in the haemolymph of S. littoralis larvae. Based on these results, senecionine [*80]N-o~ide was fed to C. transiens larvae and Z. variegatus adults. The senecionine Noxide recovered from the haemolymph of the two insects shows an almost complete loss of "0 label, indicating reduction of the orally fed N-oxide in the guts, uptake of the tertiary alkaloid and its re-N-oxidation in the haemolymph. The enzyme responsible for N-oxidation is a soluble mixed function monooxygenase. It was isolated from the haemolymph of the sequestering arctiid Tyria jacobaeue and purified to electrophoretic homogeneity. The enzyme is a flavoprotein with a native M , of 200000 and a subunit M , of 51 000. It shows a pH optimum at 7.0, has its maximal activity at a temperature of 40-45°C and an isoelectric point at pH 4.9. The reaction is strictly NADPH-dependent (K,,, 1.3 pM). From 20 pyrrolizidine alkaloids so far tested as substrates, the enyzme N-oxidizes only alkaloids with structural elements which are essential for hepatotoxic and genotoxic pyrrolizidine alkaloids (i.e. 1,2-double bond, esterification of the allylic hydroxyl group, presence of a second free or esterified hydroxyl group at carbon 7). A great variety of related alkaloids and xenobiotics were tested as substrate, none was accepted. The K, values of senecionine, monocrotaline and heliotrine, representing the three main types of pyrrolizidine alkaloids, are 1.3 pM, 12.5 pM and 290 pM, respectively. The novel enzyme was named senecionine N-oxygenase (SNO). The enzyme was partially purified from two other arctiids. The three SNOs show the same general substrate specificity but differ in their affinities towards the main structural types of pyrrolizidine alkaloids. The enzymes from the two generalists (Creatonotos transiens and Arctia caja) display a broader substrate affinity than the enzyme from the specialist (Tyria jacobaeae). The two molecular forms of pyrrolizidine alkaloids, the lipophilic protoxic tertiary amine and its hydrophilic nontoxic N-oxide are discussed in respect to their bioactivation and detoxification in mammals and their role as defensive chemicals in specialized insects. Pyrrolizidine-alkaloid-sequestering insects store the alkaloids as nontoxic N-oxides which are reduced in the guts of any potential insectivore. The lipophilic tertiary alkaloid is absorbed passively and then bioactivated by cytochrome P-450 oxidase.Keywords: Tyria jacobaeae (Lepidoptera, Arctiidae) ; pyrrolizidine alkaloid; alkaloid uptake; senecionine N-oxygenase ; chemical defense.Pyrrolizidine alkaloids are unique among the some 20 000 p...

Evolutionary recruitment of a flavin-dependent monooxygenase for the detoxification of host plant-acquired pyrrolizidine alkaloids in the alkaloid-defended arctiid mothTyriajacobaeae

“…SNO purified from T. jacobaeae shows two distinct bands after SDS͞PAGE (21). Both were subjected to microsequencing, resulting in one Nterminal peptide of 26 amino acids (pep1).…”

“…H]atropine (87 Ci͞mmol) was purchased from DuPont. Pyrrolizidine alkaloids applied as enzyme substrates were obtained as described (21).…”

“…SNO was purified from hemolymph of T. jacobaeae as described (21). The purified enzyme was transferred to a poly(vinylidene difluoride) membrane (Millipore) and was subjected to microsequencing.…”

“…Heterologous expression of the SNO of T. jacobaeae in E. coli BL21(DE3) resulted in the formation of inclusion bodies, although the native enzyme is a soluble protein (21). Attempts to avoid the formation of inclusion bodies by varying the culture conditions (temperature for growth and induction, concentration of IPTG) were unsuccessful.…”

“…N-oxidation is only one of the different, recently identified strategies insects developed in evolution to safely handle plant-acquired PAs for their own defense (22,23). The enzyme responsible for the N-oxidation of tertiary PAs in Tyria is senecionine N-oxygenase (SNO), which is a reduced nicotinamideadenine dinucleotide phosphate (NADPH)-dependent flavin monooxygenase (FMO), not a cytochrome P450 enzyme (21). In contrast to the intensively studied mammalian multisubstrate FMOs (24), the Tyria enzyme is strictly substrate specific.…”