Fatty acid 9- and 13-hydroperoxide lyases from cucumber.
Fatty acid hydroperoxide lyase (HPL) is a novel P-450 enzyme that cleaves fatty acid hydroperoxides to form short-chain aldehydes and oxo-acids. In cucumber seedlings, the activities of both fatty acid 9HPL and 13HPL could be detected. High 9HPL activity was especially evident in hypocotyls. Using a polymerase chain reaction-based cloning strategy, we isolated two HPL-related cDNAs from cucumber hypocotyls. One of them, C17, had a frameshift and it was apparently expressed from a pseudogene. After repairing the frameshift, the cDNA was successfully expressed in Escherichia coli as an active HPL with specificity for 13-hydroperoxides. The other clone, C15, showed higher sequence similarity to allene oxide synthase (AOS). This cDNA was also expressed in E. coli, and the recombinant enzyme was shown to act both on 9- and 13-hydroperoxides, with a preference for the former. By extensive product analyses, it was determined that the recombinant C15 enzyme has only HPL activity and no AOS activity, in spite of its higher sequence similarity to AOS.
Matsui
K
K
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan. matsui@agr.yamaguchi-u.ac.jp
Ujita
C
C
Fujimoto
S
S
Wilkinson
J
J
Hiatt
B
B
Knauf
V
V
Kajiwara
T
T
Feussner
I
I
D000446:Aldehyde-Lyases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000447:Aldehydes / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D018553:Cucumis sativus / Q000201:enzymology*; D003577:Cytochrome P-450 Enzyme System; D016368:Frameshift Mutation / Q000235:genetics; D006836:Hydro-Lyases; D006861:Hydrogen Peroxide / Q000378:metabolism; D018546:Hypocotyl / Q000201:enzymology; D019746:Intramolecular Oxidoreductases / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D011544:Pseudogenes / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D018749:RNA, Plant / Q000032:analysis / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D000447:Aldehydes; D012333:RNA, Messenger; D018749:RNA, Plant; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D006861:Hydrogen Peroxide; D000446:Aldehyde-Lyases; C054540:hydroperoxide lyase; C443399:9-hydroperoxide lyase; D006836:Hydro-Lyases; D019746:Intramolecular Oxidoreductases; C021377:hydroperoxide isomerase