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

Abstract

ci-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 ci-hydroxypaimitic acid. As the crude cell-free preparation also catalyzes the formation of other hydroxy acids such as a-and l8-hydroxy acids, the o-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 whydroxylation product. This enzymic hydroxylation required 02 and NADPH, but substitution of NADH resulted in nearly half the reaction rate obtained with NADPH. Maximal rates of w-hydroxylation occurred at pH 8 and the rate increased in a sigmoidal manner with increasing concentrations of palmitic acid. This w-hydroxylation was inhibited by the cassicad mixed function oxidase inhibitors such as metal chelators (ophenanthroline, 8-hydroxyquinoline, and a,a-dipyridyl), NaN3 and thiol reagents (N-ethylmaleimide andp-chloromercuribenzoate). As expected of a hydroxylase, involving cytochrome P450, the present c-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).Palmitic acid, 16-hydroxypalmitic acid, and 10,16-dihydroxypalmitic acid constitute the most common components of cutin (10,15). Exogenous labeled palmitic acid was incorporated into both hydroxy acids, whereas labeled 16-hydroxypalmitic acid was incorporated specifically into the dihydroxy acid in Vicia faba leaf discs (12). From such results we concluded that palmitic acid was hydroxylated first at the co-carbon followed by another hydroxylation at C-10. Incorporation of specifically labeled precursors into cutin and suberin in tissues slices showed that w-hydroxylation of linoleic acid and/or oleic acid was also involved in the biosynthesis of the major monomers of cutin and suberin (5,13 (soluble PVP, Mr 400,000) was provided by GAF Corp. Carbon monoxide (research grade, 99.99%) was purchased from Matheson Gas Products, while the other gases were obtained from Liquid Air, Inc. Hexadecane-1,16-dioic acid was purchased from K & K Lab., 16-hydroxyhexadecanoic acid from Aldrich Chemical Co., and 2-hydroxyhexadecanoic acid from Analabs, Inc. Hexadecane-1,16-diol and hexadecane-1,2-diol were prepared by LiAlH4 reduction of hexadecane-1,16-dioic acid and 2-hydroxyhexadecanoic acid, respectively. Both of these diol standards were purified by TLC on Silica Gel G with diethyl ether-hexane-methanol (...

Nanoridges that characterize the surface morphology of flowers require the synthesis of cutin polyester

“…It accounts, for example, for 80% of cutin in leaves of Vicia faba (37) and in fruits of tomato (38). The cutin monomer profiles of the Arabidopsis insertional mutants for CYP86A4 and CYP77A6 presented here indicate that the -hydroxylase and in-chain hydroxylase for the biosynthesis of 10,16-dihydroxypalmitate, which were studied by Kolattukudy and coworkers 30 years ago using biochemical assays in V. faba (39,40), have now been identified in Arabidopsis. Furthermore, we have confirmed that the reaction sequence of -hydroxylation followed by in-chain hydroxylation that was proposed on the basis of in vitro studies occurs in vivo (Fig.…”

Biosynthesis and function of polyacetylenes and allied natural products

“…ω-Oxidation of fatty acids by cytochrome-P 450 enzymes occurs widely and has major relevance in cutin and suberin biosynthesis [172,173], protection against postgenital organ fusion [174], and other aspects of plant development. Related CYP 450 s with specific reductant requirements have been found in Fusarium oxysporum [175].…”

Time Course of Induction of Cytochrome P-450, NADPH-Cytochrome c Reductase, and Cinnamic Acid Hydroxylase by Phenobarbital, Ethanol, Herbicides, and Manganese in Higher Plant Microsomes

“…The mixed function oxidase trans-cinnamic acid 4-hydroxylase, cytochrome P450, cytochrome b5, and NADPH-cytochrome c (P450) reductase were measured in microsomes from aging artichoke tuber slices exposed to (24,25), and trans-cinnamic acid (1, 18) are hydroxylated, whereas p-chloro-N-methylaniline (27) was N-demethylated in Cyt P-450-dependent reactions. Involvment of Cyt P-450 is also suggested in the N-dealkylation of the herbicide Monuron2 (10).…”