Abstract
A pathogen-inducible oxygenase in tobacco leaves and a homologous enzyme from Arabidopsis were recently characterized (Sanz, A., Moreno, J. I., and Castresana, C. (1998) Plant Cell 10, 1523-1537). Linolenic acid incubated at 23 degrees C with preparations containing the recombinant enzymes underwent alpha-oxidation with the formation of a chain-shortened aldehyde, i.e., 8(Z),11(Z), 14(Z)-heptadecatrienal (83%), an alpha-hydroxy acid, 2(R)-hydroxy-9(Z),12(Z),15(Z)-octadecatrienoic acid (15%), and a chain-shortened fatty acid, 8(Z),11(Z),14(Z)-heptadecatrienoic acid (2%). When incubations were performed at 0 degrees C, 2(R)-hydroperoxy-9(Z),12(Z),15(Z)-octadecatrienoic acid was obtained as the main product. An intermediary role of 2(R)-hydroperoxy-9(Z), 12(Z),15(Z)-octadecatrienoic acid in alpha-oxidation was demonstrated by re-incubation experiments, in which the hydroperoxide was converted into the same alpha-oxidation products as those formed from linolenic acid. 2(R)-Hydroperoxy-9(Z),12(Z), 15(Z)-octadecatrienoic acid was chemically unstable and had a half-life time in buffer of about 30 min at 23 degrees C. Extracts of cells expressing the recombinant oxygenases accelerated breakdown of the hydroperoxide (half-life time, about 3 min at 23 degrees C), however, this was not attributable to the recombinant enzymes since the same rate of hydroperoxide degradation was observed in the presence of control cells not expressing the enzymes. No significant discrimination between enantiomers was observed in the degradation of 2(R,S)-hydroperoxy-9(Z)-octadecenoic acid in the presence of recombinant oxygenases. A previously studied system for alpha-oxidation in cucumber was re-examined using the newly developed techniques and was found to catalyze the same conversions as those observed with the recombinant enzymes, i.e. enzymatic alpha-dioxygenation of fatty acids into 2(R)-hydroperoxides and a first order, non-stereoselective degradation of hydroperoxides into alpha-oxidation products. It was concluded that the recombinant enzymes from tobacco and Arabidopsis were both alpha-dioxygenases, and that members of this new class of enzymes catalyze the first step of alpha-oxidation in plant tissue.
Highlights
A pathogen-inducible oxygenase in tobacco leaves and a homologous enzyme from Arabidopsis were recently characterized
Incubation of Linolenic Acid with Recombinant Enzymes from Tobacco Leaves or Arabidopsis—A preparation of the tobacco leaf oxygenase obtained from insect cells infected with baculovirus carrying pFASTBAC-tob.A5.2 was stirred for 20 min at 23 °C with 100 M [9,10,12,13,15,16-3H6]linolenic acid
A pathogen-inducible oxygenase (PIOX) in tobacco leaves and a homologous enzyme from Arabidopsis were identified in recent work (22)
Summary
A pathogen-inducible oxygenase in tobacco leaves and a homologous enzyme from Arabidopsis were recently characterized I.e. the 13(S)-hydroperoxide derivative of linolenic acid, is converted into jasmonic acid by a series of reactions catalyzed by allene oxide synthase, allene oxide cyclase, reductase, and -oxidation enzymes (2) This pathway is of biological importance in plants because it produces compounds which are involved in defense reactions against insects and other phytopathogens (16), in mechanical responses such as tendril coiling (17), and pollen development (18). Tobacco leaves were found to accumulate a 75-kDa protein in response to bacterial infection (22) This protein, as well as a protein from Arabidopsis showing a 75% homology in amino acid sequence, were expressed in insect cells and found to cause uptake of molecular oxygen in the presence of polyunsaturated fatty acids such as linolenic acid, linoleic acid, and arachidonic acid. The mode of degradation of these unstable hydroperoxides into chain-shortened aldehydes and other ␣-oxidation products has been studied
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