I The oxidative pathway proceeds through formation of hydroxycinnamic acid CoA-esters and subsequent NAD-dependent oxidation of the side chain in reactions, similar to β-oxidation of fatty acids, leading from ferulic acid to vanillic acid and vanillin (Zenk 1965) and from 4-coumaric acid to 4-hydroxybenzoic acid (Loschler and Heide 1994).
II The non-oxidative pathway proceeds through a cofactor independent action of a hydrolyase type enzyme, 4-hydroxybenzaldehyde synthase (4HBS), on 4-coumaric acid producing 4-hydroxybenzaldehyde (French et al. 1976; Yazaki et al. 1991; Schnitzler et al. 1992; Podstolski et al. 2002), and on 2-coumaric acid producing salicylic aldehyde (Malinowski et al. 2007).
III The shikimate pathway proceeds via isochorismic acid producing salicylic acid (2-hydroxybenzoic acid) and 2,3-dihydroxybenzoic acid (Wildermuth et al. 2001; Muljono et al. 2002). This pathway is not dependent on phenylalanine as a precursor.
In the bacterium Pseudomonas fluorescens, there is yet another non-oxidative pathway in which feruloyl-CoA ester undergoes chain shortening by an enoyl-SCoA hydratase/isomer-ase type enzyme, ending with the respective aldehyde (vanillin) formation (Gasson et al. 1998; Mitra et al. 1999).
A very different pathway to vanillic acid biosynthesis in cell cultures of V. planifolia has been proposed by Funk and Brodelius (1990). It involves conversion of cinnamic acid glucose ester to the corresponding caffeic acid glucose ester, which then undergoes two steps of methylation, first in position 4 forming isoferulic acid and then in position 3 forming 3,4-dimethoxycinnamic acid glucose ester. The last compound then serves as a substrate for a chain-shortening enzyme requiring methylation of position 4 of hydroxycinnamic acid. As a result, 3,4-dimethoxybenzoic acid is formed, which upon demethylation in position 4 yields vanillic acid. This pathway still awaits direct enzymatic evidence.
A tempting idea presented by Zenk (1965) that vanillin in V. planifolia may be produced through the β-oxidative pathway from ferulic acid, a compound having the identical substitution pattern in the ring as does vanillin, has so far gained no support on the enzymatic side. None of the proposed enzymes of a β-oxidation-like-pathway leading from ferulic acid to vanillin have been characterized.
On the other hand, a non-oxidative chain shortening enzyme, 4HBS (Mr 28kDa), converting 4-coumaric acid to 4-hydroxybenzaldehyde was isolated, purified, and characterized from V. planifolia embryo culture (Podstolski et al. 2002). 4HBS activity was highly specific for 4-coumaric acid. Cinnamic acid or further metabolites of the phenylpropanoid pathway, 2-coumaric, caffeic and sinapic acids, were not substrates. The activity with ferulic acid was very low, about 2% of that obtained with 4-coumaric acid. 4HBS required thiol reagents for its activity. In this respect, equally effective were dithiothreitol, CoA, and to a lesser extent cysteine and the reduced form of glutathione (Podstolski et al. 2002). 4HBS catalyzed a hydrolyase type reaction. This reaction presumably proceeds via an unstable intermediate, 4-hydroxyphenyl-p-hydroxypropionic acid, formed after hydration of the side chain double bond of 4-coumaric acid followed by side chain cleavage and release of acetate and 4-hydroxybenzaldehyde (Figure 17.1) (Yazaki et al. 1991; Schnitzler et al. 1992). The corresponding activity converting 2-coumaric acid to salicylic aldehyde (2-hydroxybenzaldehyde) was characterized from tobacco (Malinowski et al. 2007).
Fig. 17.1 Proposed vanillin biosynthetic pathway (modified from Kanisawa et al. 1994 and Podstolski ef al. 2002) in Vanilla planifolia. Bold arrows indicate suggested main flow of metabolites. Abbreviations: PAL, L-phenylalanineammonia-lyase; C4H, cinnamic acid 4-hydroxylase; C3H, 4-hydroxybenzaldehyde 3-hydroxylase; 4HBS, 4-hydroxybenzaldehyde synthase; p-gluc, β-D-glucosidase; GT, glucosyltransferase; UDPG, uridine diphosphate glucose; BAD, 4-hydroxybenzoyl alcohol dehydrogenase; OMT, O-methyl-transferase; SAM, S-adenosylmethionine; NADPH, nicotinamide adenine dinucleotide phosphate, reduced; Tartrate esters: bis[4-(β-D-glucopyranosyloxy)-benzyl]-2-isopropyltartrate and bis[4-(β-D-glucopyranosyloxy)-benzyl]-2-(2-butyl)tartrate.