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Articles

Biosynthesis of Cutin

Enzymatic Conversion of -Hydroxy Fatty Acids to Dicarboxylic Acids by Cell-free Extracts of Vicia Faba Epidermis ¹

^a Department of Agricultural Chemistry and Program in Biochemistry and Biophysics, Washington State University, Pullman, Washington 99163

Long chain dicarboxylic acids are constituents of the protective biopolymers cutin and suberin of plants. Cell-free extracts from the excised epidermis of Vicia faba leaves catalyzed conversion of 16-hydroxy[G-³H]hexadecanoic acid to the corresponding dicarboxylic acid with nicotinamide-adenine dinucleotide phosphate as the preferred cofactor. This enzymatic activity, located largely in the 100,000g supernatant fraction, had a pH optimum near 8. This dehydrogenase showed an apparent Km of 1.25 x 10^–5M and 3.6 x 10^–4M for 16-hydroxyhexadecanoic acid and NADP, respectively. Modification of the substrate, either by esterification of the carboxyl group or by introduction of another hydroxyl group at C-10, resulted in a substantial (two-thirds) decrease in the rate of reaction, and hexadecanol was not a good substrate. The enzyme was inhibited by thiol reagents such as N-ethylmaleimide and p-chloromercuribenzoate. The aldehyde intermediate was trapped by the inclusion of dinitrophenyl hydrazine in the reaction mixture, and the 16-oxo compound was regenerated and identified. Furthermore, synthetic 16-oxo-[G-³H] hexadecanoic acid was readily converted to the dicarboxylic acid by the cell-free preparation. These results demonstrate that epidermis of Vicia faba contains an -hydroxyacid dehydrogenase and an -oxoacid dehydrogenase.

³ Present address: Department of Biochemistry, University College of Wales, Swansea, Wales.

¹ This work was supported in part by National Science Foundation Grants GB-23081 and GB-43076X. Scientific Paper No. 4282, project 2001, College of Agriculture Research Center, Washington State University, Pullman, Wash. 99163.