Solubilization and characterization of a senecionine N-oxygenase from Crotalaria scassellatii seedlings

Abstract

Abstract Seeds of Crotalaria scassellatii (Fabaceae) store pyrrolizidine alkaloids as tertiary amines. During the beginning of seed germination the tertiary alkaloids are rapidly converted into the respective alkaloid N -oxides which are the ultimate forms of alkaloid transport, metabolism and storage in vegetative plant organs. The enzyme catalyzing the N -oxidation was isolated from 2-day old seedling, partially purified and characterized. It is a membrane-bound, but not microsomal enzyme sedimenting in the 39 000 g fraction. The particulate enzyme was solubilized in the presence of 0.4% CHAPS and 0.4 M NaCl. The solubilized enzyme was partially purified (228-fold) by means of 70% ammonium sulfate precipitation and monocrotaline affinity chromatography. Inhibitor experiments, temperature sensitivity and lack of a carboxy ferrocytochrome absorption maximum at 450 nm strongly indicate SNO to be a flavin dependent enzyme. It is a mixed function monooxygenase that specifically N -oxidizes a number of structurally related pyrrolizidine alkaloids including the alkaloids of Crotalaria . A great variety of related alkaloids and xenobiotics were tested as substrates, none was accepted. The apparent K m values of senecionine, monocrotaline and heliotrine representing the three major types of pyrrolizidine alkaloids, are 12.4, 40.1 and 370.9 μ M, respectively. Senecionine is the best substrate, consequently the enzyme was named senecionine N -oxygenase (SNO). The substrate specificity of SNO is almost identical with that of a soluble insect SNO recently characterized from the haemolymph of arctiid larvae [Lindigkeit, R., Biller, A., Buch, M., Schiebel, H.-M., Boppre, M. and Hartmann, T., European Journal of Biochemistry , 1997, 245 , 626].