Handbook of Vanilla Science and Technology

In addition to activity in the embryo culture, 4HBS activity was also observed in leaves, stems, roots, and vanilla pods. The highest specific activity was found in the pods where it was present during the whole developmental period with a peak of activity 7 to 8 months after pollination. The peak of 4HBS activity coincided with the maximum content of 4-coumaric acid and preceded the highest accumulation of 4-hydroxybenzaldehyde that is observed 10 to 11 months post-pollination (Podstolski et al. 2002). The presence of 4HBS was confirmed (using antibodies) in the cytoplasm of secretory hair-like cells located in the inner part of Vanilla fruit. This inner part of the fruit contains 95% of the total vanillin, as well as all proposed vanillin precursors, 4-coumaric acid, 4-hydroxybenzaldehyde and 3,4-dihydrox-ybenzaldehyde (Joel et al. 2003).

17.3 4-COUMARIC ACID 3-HYDROXYLASE (C3H)

In the proposed sequence of reactions leading to vanillin (Figure 17.1), the step following 4-hydroxybenzaldehyde formation is its hydroxylation in position 3 to protocatechuic aldehyde (3,4-dihydroxybenzaldehyde). This step involves introduction of the second hydroxyl group in the position ortho to the already existing one. Enzymes catalyzing such reactions, for example 4-coumarate 3-hydroxylase (C3H), which catalyzes formation of 3,4-dihydroxycinnamic acid (caffeic acid) from 4-coumaric acid, have not been fully characterized. It has been proposed that this hydroxylation might be carried out by monophenolase activity in the presence of an electron donor. The activity of a mono-phenolase oxidizing 4-coumaric acid, 4-hydroxybenzyl alcohol, 4-hydroxybenzaldehyde, and 4-hydroxybenzoic acid was found in V. planifolia shoot primordial culture (Debowska and Podstolski 2001), but its role in formation of 3,4-dihydroxy-derivatives was not confirmed. C3H has been characterized as a mixed function oxidase containing copper in the active site (Vaughan and Butt 1970) and requiring for its activity as electron donors either ascorbate (Kojima and Tekeuchi 1989), or NADPH (Vaughan and Butt 1970), or when in the chloroplast, plastoquinone or ferredoxin (Bartlett et al. 1972). On the other hand, there is data showing that phenolase and 3-hydroxylase activities could be completely separated, giving strong evidence for the existence of two independent enzymes (Duke and Vaughn 1982). More recent data obtained with Arabidopsis (Nair et al. 2002) showed that in this model plant cytochrome P450 monooxygenase, encoded by CYP98A3, was capable of 3-hydroxylation of 4-coumaric acid. This evidence strongly supports the view that C3H is a cytochrome P450, NADPH-dependent type monooxygenase, rather than a phenolase.

There is no available data concerning plant 4-hydroxybenzaldehyde 3-hydroxylase or 4-hydroxybenzoic acid 3-hydroxylase activities. Nevertheless, such activity (preferring NADPH to NADH as a co-substrate) was described in the bacterium Corynebacterium glutamicum growing on 4-hydroxybenzoic acid as the sole carbon source (Huang et al. 2008). This finding may create an opportunity for transgenic plant engineering.

17.4 O-METHYLTRANSFERASE (OMT)

Protocatechuic aldehyde (3,4-dihydroxybenzaldehyde) is believed to be the immediate precursor of vanillin. Occurrence of this compound has been confirmed in vanilla pods (Joel et al. 2003) and in Vanilla tissue cultures as well (Havkin-Frenkel et al. 1996). Methylation of the hydroxyl group at position 3 catalyzed by an O-methltransferase (OMT) is considered the final step in the biosynthesis of vanillin (3-methoxy-4-hydroxybenzalde-hyde). Reactions catalyzed by OMTs are very common in plants. These enzymes catalyze the transfer of the methyl group from S-adenosyl-L-methionine (SAM) to a hydroxyl group of various substrates.