Biosynthesis of hydroxyfatty acid polymers: enzymatic epoxidation of 18-hydroxyoleic acid to 18-hydroxy-cis-9,10-epoxystearic acid by a particulate preparation from spinach (Spinacia oleracea)

The major component of the cuticular hydroxyfatty acid polymer, cutin, of spinach leaf was identified as 18-hydroxy-cis-9,10-epoxystearic acid. A 3000g particulate fraction obtained from the homogenates of young spinach leaves catalyzed the epoxidation of 18-hydroxyoleic acid to 18-hydroxy-cis-9,10-epoxystearic acid. The epoxy acid was identified by thin-layer chromatography of the product of hydration of the oxirane function and subsequent reduction (octadecane-1,9,10,18-tetraol), on silica gel G and boric acid impregnated silica gel, and by radio gas-liquid chromatography of the periodate oxidation product of the tetraol derived from the epoxy acid. This enzymatic epoxidation required ATP, CoA, NADPH, and O2 for maximal activity. The pH optimum for epoxidation was 9.0, and the epoxidase activity was located mainly in the 3000g particulate fraction, which contained the cuticular membranes. The rate of epoxidation of the unsaturated acid was linear up to 2 h and up to a protein concentration of 1.5 mg/ml. The apparent Km and V of the enzyme was 7.5 X 10(-5) M and 180 nmol/l-h-mg protein, respectively. That the epoxidase has a stringent substrate specificity was indicated by the observation that 18-hydroxyelaidic acid, 18-acetoxyoleic acid, and oleic acid were not readily epoxidized by this enzyme preparation. Light reversible inhibition by CO suggested the involvement of cytochrome P-450 in the epoxidation. Chelators inhibited epoxidation, and this inhibition was reversed by FeSO4. The epoxidase was inhibited 30% by 0.5 mM NaCN, whereas the same concentration of NaN3 showed very little inhibition. The present epoxidase is unique in that it is associated with the 3000g particles which contain the cutin polymer and in that it requires activation of the carboxyl group distal to the epoxidation site. Enzyme preparations capable of epoxidizing 18-hydroxyoleic acid were also obtained from the skin of apple fruit and from the epidermis of Senecio odoris leaves. This is the first report on an enzyme involved in the biosynthesis of the C18 family of cutin monomer acids.