Hydroperoxide lyases (CYP74C and CYP74B) catalyze the homolytic isomerization of fatty acid hydroperoxides into hemiacetals

The conversion of linoleic acid 9-hydroperoxide (9-HPOD) by recombinant melon ( Cucumis melo L.) hydroperoxide lyase (HPL, CYP74C subfamily) was studied. Short (5 s–1 min) incubations at 0 °C followed by rapid extraction and trimethylsilylation made it possible to trap a new unstable ( t 1/2 < 30 s) product, i.e. the hemiacetal (1′ E,3′ Z)-9-hydroxy-9-(1′,3′-nonadienyloxy)-nonanoic acid. Identification was performed by GC-MS analysis and substantiated by the formation of trimethylsilyl 9-trimethylsilyloxy-9-nonyloxy-nonanoate upon catalytic hydrogenation and by 2H-labelling experiments. Both 18O atoms of [ 18O 2-hydroperoxy]9-HPOD were incorporated into the hemiacetal. Along with the hemiacetal, three chain-cleavage products, i.e. the enol (1 E,3 Z)-nonadienol and the hydrates of 3( Z)-nonenal and 9-oxononanoic acid, were trapped as their trimethylsilyl derivatives. The kinetics of 18O incorporation from [ 18O 2]9-HPOD provided strong evidence that the cleavage products originated in the hemiacetal. Linolenic and linoleic acid 13-hydroperoxides served as substrates for recombinant HPLs of melon, alfalfa ( Medicago sativa) and guava ( Psidium guajava), and in each case hemiacetals and enols were detectable by the trapping technique. The data obtained demonstrated that CYP74C and CYP74B HPLs act as isomerases performing a homolytic rearrangement of fatty acid hydroperoxides into short-lived hemiacetals which upon decomposition produce 3( Z)-nonenal, 3( Z)-hexenal and other short chain aldehydes.