title: Characterisation of recombinant Hevea brasiliensis allene oxide synthase: effects of cycloxygenase inhibitors, lipoxygenase inhibitors and salicylates on enzyme activity.
abstract: Mechanical wounding and jasmonic acid (JA) treatment have been shown to be important factors in controlling laticifer differentiation in Hevea brasiliensis (rubber tree). With the long-term aim of potentially modifying the endogenous levels of JA in H. brasiliensis by gene transfer, we describe in this paper the molecular cloning of a H. brasiliensis allene oxide synthase (AOS) cDNA and biochemical characterisation of the recombinant AOS (His(6)-HbAOS) enzyme. The AOS cDNA encodes a protein with the expected motifs present in CYP74A sub-group of the cytochrome P450 super-family of enzymes that metabolise 13-hydroperoxylinolenic acid (13-HPOT), the intermediate involved in JA synthesis. The recombinant H. brasiliensis AOS enzyme was estimated to have a high binding affinity for 13-HPOT with a K(m) value of 4.02+/-0.64 microM. Consistent with previous studies, mammalian cycloxygenase (COX) and lipoxygenase (LOX) inhibitors were shown to significantly reduce His(6)-HbAOS enzyme activity. Although JA had no effect on His(6)-HbAOS, salicylic acid (SA) was shown to significantly inhibit the recombinant AOS enzyme activity in a dose dependent manner. Moreover, it was demonstrated that SA, and various analogues of SA, acted as competitive inhibitors of His(6)-HbAOS when 13-HPOT was used as substrate. We speculate that this effect of salicylates on AOS activity may be important in cross-talking between the SA and JA signalling pathways in plants during biotic/abiotic stress.
authors.affiliation: Division of Biochemistry and Microbiology, School of Life Sciences, University of Hertfordshire, Hatfield, Herts AL10 9AB, UK.
authors.forename: Gareth
authors.initials: G
authors.lastname: Norton
authors.forename: Arokiaraj
authors.initials: A
authors.lastname: Pappusamy
authors.forename: Faridah
authors.initials: F
authors.lastname: Yusof
authors.forename: Valérie
authors.initials: V
authors.lastname: Pujade-Renaud
authors.forename: Mark
authors.initials: M
authors.lastname: Perkins
authors.forename: David
authors.initials: D
authors.lastname: Griffiths
authors.forename: Heddwyn
authors.initials: H
authors.lastname: Jones
mesh_terms: D000595:Amino Acid Sequence; D016861:Cyclooxygenase Inhibitors / Q000737:chemistry*; D003517:Cyclopentanes / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D028482:Hevea / Q000378:metabolism*; D019746:Intramolecular Oxidoreductases / Q000737:chemistry*; D007700:Kinetics; D008084:Lipoxygenase / Q000378:metabolism*; D008969:Molecular Sequence Data; D054883:Oxylipins; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry; D012459:Salicylates / Q000378:metabolism*; D015398:Signal Transduction; D017962:alpha-Linolenic Acid / Q000737:chemistry
chemical_list: D016861:Cyclooxygenase Inhibitors; D003517:Cyclopentanes; D054883:Oxylipins; D011994:Recombinant Proteins; D012459:Salicylates; D017962:alpha-Linolenic Acid; C011006:jasmonic acid; D003577:Cytochrome P-450 Enzyme System; D008084:Lipoxygenase; D019746:Intramolecular Oxidoreductases; C021377:hydroperoxide isomerase