Biosynthesis of hydroxyfatty acid polymers: Enzymatic hydration of 18-hydroxy- cis-9,10-epoxystearic acid to threo-9,10,18-trihydroxystearic acid by a particulate preparation from apple ( Malus pumila)

Abstract

A 3000 g participate fraction prepared from homogenates of the skin of young apple fruit catalyzed the hydration of 18-hydroxy- cis-9,10-epoxystearic acid to threo-9,10,18-trihydroxystearic acid, a key step in the biosynthesis of cutin, the hydroxyfatty acid biopolymer of the plant cuticle. Similar preparations from the internal tissue of apple fruit contained less than 6% of this epoxide hydrase activity. The enzymatic product was identified by thin-layer chromatography on silica gel G and on boric acid-silica gel G, as well as by radio gas-liquid chromatography of its periodate oxidation product. The epoxide hydrase required no cofactors, had a pH optimum of 6.5, and it was located mainly (>90%) in the 3000 g particle fraction which contained the cuticular membranes. The rate of hydration of the epoxy acid was linear up to 75 min and up to a protein concentration of 0.8 mg/ml. The apparent K m and V of the enzyme was 4 × 10 −4 m and 170 μmol/1-h-mg of protein, respectively. p-Chloromercuribenzoate strongly inhibited the enzyme (80% inhibition at 10 −4 m), while iodoacetamide and N-ethylmaleimide were less potent inhibitors. 1,1,1-Trichloropropylene-2,3-oxide at 5 × 10 −3 m gave 60% inhibition of hydration of 18-hydroxy-9,10-epoxystearic acid. Diisopropylfluorophosphate was mildly inhibitory, and the enzyme was insensitive to ethylenediaminetetraacetic acid. Methyl 18-hydroxy-9,10-epoxystearate, 18-acetoxy-9,10-epoxystearic acid, methyl 18-acetoxy-9,10-epoxystearate, 9,10-epoxystearic acid, and styrene oxide were not readily hydrated by this plant epoxide hydrase showing that it has a fairly stringent substrate specificity, unlike the catabolic epoxide hydrases of animals. Particulate preparations capable of hydrating 18-hydroxy-9,10-epoxystearic acid to the trihydroxy acid were also obtained from Senecio odoris leaf epidermis and Spinacia oleracea leaves. This is the first report on the enzyme which catalyzes this important step in the biosynthesis of the protective biopolymer, cutin.