Ill. Conversion of 4-Methoxycinnamic Acids into 4-Hydroxybenzoic Acids

Feeding of 4-methoxycinnamic acid, 3,4-dimethoxycinnamic acid and 3,4,5-trimethoxycinnamic acid to cell suspension cultures of Vanilla planifolia resulted in the formation of 4-hydroxybenzoic acid, vanillic acid, and syringic acid, respectively. The homologous 4-methoxybenzoic acids were demethylated to the same products. It is concluded that the side chain degrading enzyme system accepts the 4-methoxylated substrates while the demethylation occurs at the benzoic acid level. The demethylating enzyme is specific for the 4-position. Feeding of [10_4Cmethyl]-3,4-dimethoxycinnamic acid revealed that the first step in the conversion is the glycosylation of the cinnamic acid to its glucose ester. A partial purification of a UDP-glucose: transcinnamic acid glucosyltransferase is reported. 4-Methoxy substituted cinnamic acids are better substrates for this enzyme than 4-hydroxy substituted cinnamic acid. It is suggested that 4-methoxy substituted cinnamic acids are intermediates in the biosynthetic conversion of cinnamic acids to benzoic acids in cells of V. planifolia. Inhibition of 4CL4, an important enzyme of lignin biosynthesis, under in vivo conditions by 3,4-(methylenedioxy)cinnamic acid resulted in a considerably reduced formation of ligneous material with a simultaneous formation of significant amounts of vanillic acid (3). An enzymatic system converting cinnamic acids into benzoic acids was shown to be present within the cultivated cells. Furthermore, it was concluded that 4CL and CoA-esters most likely are not involved in the biosynthesis of vanillic acid (3). The fact that the cell culture produces vanillic acid under certain conditions offers a tool to study the enzymes involved in the biosynthesis of this C6-C1 compound. In our search for an efficient 4CL inhibitor 4-methoxycinnamic acid and 3,4-dimethoxycinnamic acid were also tested (3). Addition of these cinnamic acids to suspension cultures of V. planifolia resulted in the formation of 4-hydroxybenzoic acid and vanillic acid, respectively. This observation was a first indication that 4-methoxy substituted cinnamic acids are metabolized by the cells and that they may be intermediates in the biosynthesis of C6-CI compounds. In the present paper we report on extended studies on the possible role of 4methoxycinnamic acids as intermediates in the biosynthesis of 4-hydroxybenzoic acids in cell cultures of V. planifolia. Studies on the biosynthetic pathway leading from C6-C3 compounds of the general phenylpropanoid pathway (i.e. cinnamic acids) to C6-C1 compounds (i.e. benzoic acids) in cells of higher plants have recently been initiated in our laboratory. To this end, cell suspension cultures of Vanilla planifolia Andr. have been initiated (2). No C6-CI compounds could, however, be isolated from this culture under normal growth conditions (2). Elicitation of the cell culture with chitosan resulted in an activation of the general phenylpropanoid metabolism (i.e. induction of various enzymes of this pathway) leading to an increased formation of ligneous cell wall materials (2). Supported by a research grant from Danisco Biotechnology, Glostrup, Denmark. This paper is number III in a series "Phenylpropanoid metabolism in suspension cultures of Vanilla planifolia Andr. " The preceding paper is Funk and Brodelius (3) 2 Present address: Institute of Biological Chemistry Washington State University, Pullman WA 99169-6340. 3 Present address: Department of Plant Biochemistry, University of Lund, P.O. Box 7007, S-22007 Lund, Sweden. MATERIALS AND METHODS