Biosynthesis of Cutin ω-Hydroxylation of Fatty Acids by a Microsomal Preparation from Germinating Vicia faba

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Abstract
ω-Hydroxylation of fatty acids, which is a key reaction in the biosynthesis of cutin and suberin, has been demonstrated for the first time in a cell-free preparation from a higher plant. A crude microsomal fraction (105,000g pellet) from germinating embryonic shoots of Vicia faba catalyzed the conversion of palmitic acid to ω-hydroxypalmitic acid. As the crude cell-free preparation also catalyzes the formation of other hydroxy acids such as α- and β-hydroxy acids, the ω-hydroxylation product was identified by gas chromatography on a polyester column and reverse phase, high performance liquid chromatography, two techniques which were shown to resolve the positional isomers. Gas chromatographic analysis of the dicarboxylic acid obtained by CrO3 oxidation of the enzymic product also confirmed the identity of the enzymic ω-hydroxylation product. This enzymic hydroxylation required O2 and NADPH, but substitution of NADH resulted in nearly half the reaction rate obtained with NADPH. Maximal rates of ω-hydroxylation occurred at pH 8 and the rate increased in a sigmoidal manner with increasing concentrations of palmitic acid. This ω-hydroxylation was inhibited by the classical mixed function oxidase inhibitors such as metal chelators (o-phenanthroline, 8-hydroxyquinoline, and α,α-dipyridyl), NaN3 and thiol reagents (N-ethylmaleimide and p-chloromercuribenzoate). As expected of a hydroxylase, involving cytochrome P450, the present ω-hydroxylase was inhibited by CO and this enzyme system showed unusually high sensitivity to this inhibition; 10% CO caused inhibition and 30% CO completely inhibited the reaction. Another unusual feature was that the inhibition caused by any level of CO could not be reversed by light (420-460 nm).
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