Cloning, molecular and functional characterization of Arabidopsis thaliana allene oxide synthase (CYP 74), the first enzyme of the octadecanoid pathway to jasmonates.
Allene oxide synthase, an enzyme of the octadecanoid pathway to jasmonates, was cloned from Arabidopsis thaliana as a full-length cDNA encoding a polypeptide of 517 amino acids with a calculated molecular mass of 58705 Da. From the sequence, an N-terminal transit peptide of 21 amino acids resembling chloroplast transit peptides was deduced. Three out of four invariant amino acid residues of cytochrome P450 heme-binding domains are conserved and properly positioned in the enzyme coding region, including the heme-accepting cysteine (Cys-470). Southern analysis indicated in A. thaliana only one allene oxide synthase gene to be present. While transcript levels were rapidly and transiently induced after wounding of the leaves, allene oxide synthase activity remained nearly constant at a low level of ca. 0.8 nkat per mg of protein. The cDNA encoding A. thaliana allene oxide synthase was highly expressed in bacteria giving rise to a polypeptide of the calculated molecular mass. The protein was enzymatically active, and verification of the reaction products by GC-MS showed that it was capable of utilizing not only 13-hydroperoxylinolenic acid (13-hydroperoxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid), but also 13-hydroperoxylinoleic acid (13-hydroperoxy-9(Z), 11(E)-octadecadienoic acid) as substrate. The data suggest parallel pathways to jasmonates from linolenic acid or linoleic acid in A. thaliana.
Laudert
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Lehrstuhl für Pflanzenphysiologie, Ruhr-Universität Bochum, Germany.
Pfannschmidt
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U
Lottspeich
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F
Holländer-Czytko
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Weiler
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EW
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D003517:Cyclopentanes / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D017343:Genes, Plant; D019746:Intramolecular Oxidoreductases; D007535:Isomerases / Q000235:genetics*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D054883:Oxylipins; D012333:RNA, Messenger / Q000235:genetics; D018749:RNA, Plant / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D003517:Cyclopentanes; D017931:DNA Primers; D018076:DNA, Complementary; D054883:Oxylipins; D012333:RNA, Messenger; D018749:RNA, Plant; D011994:Recombinant Proteins; C011006:jasmonic acid; D007535:Isomerases; D019746:Intramolecular Oxidoreductases; C021377:hydroperoxide isomerase