Ferulic acid hydroxylase-1

Coniferaldehyde (3.76) can undergo several fates, some of which can ultimately lead to the same end product. It can be reduced to coniferyl alcohol (3.79) by the enzyme cinnamyl alcohol dehydrogenase (CAD). Alternatively, the enzyme coniferyl aldehyde/coniferyl alcohol 5-hydroxylase (C5H), also known by its less accurate name ferulic acid 5-hydroxylase (F5H Humphreys et al., 1999) can catalyze the hydroxylation of C5 to result in 5-hydroxyconiferyl aldehyde (3.77). C5H is also able to form 5-hydroxyconiferyl alcohol (3.80) from coniferyl alcohol (3.79). This enzyme was initially identified as F5H, after analysis of the Arabidopsis ferulic acid hydroxylase 1 (fahl) mutant, which was isolated in a mutant screen based on reduced levels of the UV-fluorescent sinapoyl esters (Section 13 Chappie et al., 1992). The FAH1 gene was cloned using a T-DNA tagged mutant allele (Meyer et al., 1996), which revealed that the... 

A cytochrome P450 with ferulic acid 5-hydroxylase (F5H) activity was first detected by Grand (1984) in poplar. The gene for this cytochrome P450 defining the new CYP84 family was described and isolated with the help of an... 

Grand, C. (1984) Ferulic acid 5-hydroxylase a new cytochrome P4so-dependent enzyme from higher plant microsomes involved in lignin synthesis. FEBS Lett., 169, 7-11. 

GRAND, C., Ferulic Acid 5-Hydroxylase - a New Cytochrome P-450-Dependent Enzyme from Higher-Plant Microsomes Involved in Lignin Synthesis, FEBS Lett., 1984,169, 7-11. 

Figure 3-4. The general phenylpropanoid pathway. The enzymes involved in this pathway are (a) phenylalanine ammonia lyase (PAL E.C. 4.3.1.5), (b) cinnamic acid 4-hydroxylase (C4H E.C. 1.14.13.11), and (J) 4-coumaric acid CoA ligase (4CL E.C. 6.2.1.12). (a) depicts tyrosine ammonia lyase activity in PAL of graminaceous species. The grey structures in the box represent an older version of the phenylpropanoid pathway in which the ring substitution reactions were thought to occur at the level of the hydroxycinnamic acids and/or hydroxycinnamoyl esters. The enzymes involved in these conversions are (c) coumarate 3-hydroxylase (C3H E.C. 1.14.14.1), (d) caffeate O-methyltransferase (COMT EC 2.1.1.68), (e) ferulate 5-hydroxylase (F5H EC 1.14.13), and (g) caffeoyl-CoA O-methyltransferase (CCoA-OMT EC 2.1.1.104). These enzymes are discussed in more detail in Section 10.

Figure 1.35 Schematic diagram of the phenolic biosynthetic pathway accompanied by the key enzymes involved. Enzyme abbreviations PAL, phenylalanine ammonia-lyase BA2H, benzoic acid 2-hydroxylase C4H, cinnamate 4-hydroxylase COMT-1, caffeic/5-hydroxyferulic acid O-methy I transferase 4CL, p-co um a ra te C o A ligase F5H, ferulate 5-hydroxylase GT, galloyltransferase ACoAC, acetylCoA carboxylase.

Synthesis of caffeic and ferulic acids also needs hydroxylase and methyltransferase enzymes transformation into hydroxycinnamic tartaric acid esters (HCTA) is operated by an acyltransferase enzyme. The scheme shown in Figure 2.9 summarizes the biosynthetic pathways described. 

L-Phenylalanine,which is derived via the shikimic acid pathway,is an important precursor for aromatic aroma components. This amino acid can be transformed into phe-nylpyruvate by transamination and by subsequent decarboxylation to 2-phenylacetyl-CoA in an analogous reaction as discussed for leucine and valine. 2-Phenylacetyl-CoA is converted into esters of a variety of alcohols or reduced to 2-phenylethanol and transformed into 2-phenyl-ethyl esters. The end products of phenylalanine catabolism are fumaric acid and acetoacetate which are further metabolized by the TCA-cycle. Phenylalanine ammonia lyase converts the amino acid into cinnamic acid, the key intermediate of phenylpropanoid metabolism. By a series of enzymes (cinnamate-4-hydroxylase, p-coumarate 3-hydroxylase, catechol O-methyltransferase and ferulate 5-hydroxylase) cinnamic acid is transformed into p-couma-ric-, caffeic-, ferulic-, 5-hydroxyferulic- and sinapic acids,which act as precursors for flavor components and are important intermediates in the biosynthesis of fla-vonoides, lignins, etc. Reduction of cinnamic acids to aldehydes and alcohols by cinnamoyl-CoA NADPH-oxido-reductase and cinnamoyl-alcohol-dehydrogenase form important flavor compounds such as cinnamic aldehyde, cin-namyl alcohol and esters. Further reduction of cinnamyl alcohols lead to propenyl- and allylphenols such as... 

Figure 4 Current view of the phenylpropanoid pathway to the monolignols 19-23. 4CL, 4-hydroxycinnamate coenzyme Aligases pC3H , p-coumarate 3-hydroxylase C4H, cinnamate 4-hydroxylase CAD, cinnamyl alcohol dehydrogenases CCOMT, hydroxycinnamoyl CoA O-methyltransferases CCR, cinnamoyl-CoA oxidoreductases COMT, caffeic acid O-methyltransferases F5H , ferulate 5-hydroxylase HCT, hydroxycinnamoyl-CoA shikimate hydroxycinnamoyltransferase HOT, hydroxycinnamoyl-CoA quinate hydroxycinnamoyltransferase PAL, phenylalanine ammonia lyase TAL, tyrosine ammonia lyase.

Ferulate 5-hydroxylase (F5H) catalyzes the third P-450 hydroxylation step at the C5 position of the phenolic ring to ultimately afford, via the monolignol pathway, 5-hydroxyconiferyl (22) and/or sinapyl (23) alcohols. This P-450 was first detected in xylem and sclerenchyma-enriched tissues from poplar Populus x euramericana), with microsomal extracts able to catalyze hydroxylation of ferulic acid (6) to 5-hydroxyferulic acid (7) in the presence of NADPH with apparent values of 6.3 pmoll / This enzyme was thus characterized as F5H. 

Figure 3.1 Primary flux of carbon through phenylpropanoid pathway in Arabidopsis. PAL, phenylalanine ammonia-lyase 4CL, 4-(hydroxy)cinnamoyl CoA ligase C4H, cinnamate 4-hydroxylase HCT, hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase C3 H, /7-coumaroylshikimate 3 -hydroxylase CCoAOMT, caffeoyl CoA O-methyltransferase F5H, ferulate 5-hydroxylase COMT, caffeic acid/5-hydroxyferulic acid o-methyltransferase CCR, cinnamoyl CoA reductase CAD, cinnamyl alcohol dehydrogenase. Not depicted is the HCT catalyzed synthesis of/r-coumaroyl quinate.

Figure 3. Proposed pathways to AA precursors. A) 3,4-dihydroxybenzaldehyde (3,4-DHBA) biosynthesis depicting the two possible routes from/ -coumaric acid to form 3,4-DHBA the oxidative ferulate and the non-oxidative benzoate pathways B) Tyramine biosynthesis. Arrows without labeling reflect chemical reactions that have not been enzymatically characterized. Enzymes that have been cloned, characterized and identified are labeled in black bold. Enzyme abbreviations PAL, phenylalanine ammonia -lyase C4H, cinnamate 4-hydroxylase C3H, coumarate 3-hydroxylase HBS, 4-hydroxybenzaldehyde synthase TYDC, tyrosine decarboxylase.

Some relatively nonspecific enzymatic formation of caffeic (12), ferulic (13), and synapic (14) acids has been noted (Davin et al., 1992). Monooxygenases of microsomal fractions appear to be involved. For example, a specific p-coum-arate-3-hydroxylase has been isolated from mung beans. However, other work suggests that the carboxyl group of p-coumaric acid must be esterified as a quinic acid ester before... 

---