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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. ß

Fatty acid hydroperoxide iyases cleave a C-C bond adjacent to a hydroperoxide group in lipoxygenase derived lipid hydroperoxides to form short-chain aldehydes and oxo-acids. Previously, we showed that fatty acid hydroperoxide iyase from bell pepper fruits is a heine protein whose spectrophotometric properties greatly resemble a cytochrome P450. In order to ascertain the relationship of it to the P450 gene family, we have cloned cDNA encoding fatty acid hydroperoxide lyase from immature bell pepper fruits. The cDNA encodes 480 amino acids, and shares homology with P450s mostly at the C terminus. The heme binding cysteine is recognizable at position 441. The most closely related P450 is allene oxide synthase (CYP74A), with which it has 40% identity. It qualifies the lyase as a member of a new P450 subfamily, CYP74B. From this finding, the enzyme is thought to be a novel member of P450 specialized for the metabolism of lipid peroxides.

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