Enzymes cinnamic acid hydroxylase

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... 

Two isozymes of PAL were reported earlier in Quercus pedunculata leaves, one form being sensitive to repression by hydroxycinnamate and the other to repression by hydroxybenzoate (see last year s Report ). These two isozymes have now been isolated from the roots of the same plant and a study of their localization indicates that the cinnamate-sensitive isozyme is present in the microsomal fraction, whereas the benzoate-sensitive isozyme is present in the mitochondrial and microbody fraction. The distributions of the two forms of PAL are correlated with those of two other enzymes of phenolic biosynthesis. The enzyme cinnamate 4-hydroxylase accompanies the first isozyme in the microbodies whereas benzoate synthetase is present with the second in the microbodies. Thus phenolic acid biosynthesis is closely comjrartmented in the oak plant, Cg—C3 acids being produced exclusively in the microsomes and Cs—C, acids in the microbodies. 

The chemical and physical evidence for the presence of lignin in the material deposited at wound margins is supported by biochemical studies on the enzymes involved in phenylpropanoid metabolism. Thus, the extractable activities of phenylalanine ammonia-lyase, tyrosine ammonia-lyase, cinnamate-4-hydroxylase, caffeic acid O-methyltransferase,... 

A different approach to investigate active lignification during resistance reactions is provided by the determination of enzyme activities involved in lignin biosynthesis. Resistant plants are expected to be more strongly activated during or immediately preceding the resistance reaction compared to susceptible plants. Thus, phenylalanine ammonia-lyase (PAL) (43-45), cinnamic acid 4-hydroxylase (46), O-methyltransferases (44), and... 

Coumaroyl-CoA is produced from the amino acid phenylalanine by what has been termed the general phenylpropanoid pathway, through three enzymatic conversions catalyzed by phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumarate CoA ligase (4CL). Malonyl-CoA is formed from acetyl-CoA by acetyl-CoA carboxylase (ACC) (Figure 3.2). Acetyl-CoA may be produced in mitochondria, plastids, peroxisomes, and the cytosol by a variety of routes. It is the cytosolic acetyl-CoA that is used for flavonoid biosynthesis, and it is produced by the multiple subunit enzyme ATP-citrate lyase that converts citrate, ATP, and Co-A to acetyl-CoA, oxaloacetate, ADP, and inorganic phosphate. ... 

Fig. (2). Proposed pathways of SA and 4HBA biosynthesis. Enzymatic steps for which the enzymes have been identified include PAL, CA4H (cinnamic acid 4-hydroxylase), and BA2H (benzoic acid 2-hydroxylase). Taken from Smith-Becker et al., 1998, [55].

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.

Reichhart, D., Simon, A., Durst, F., Mathews, J.M., and Ortiz de Montellano, P.R., Autocatalytic inactivation of plant cytochrome P-450 enzymes selective inactivation of cinnamic acid 4-hydroxylase from Helianthus tuberosus by 1-aminobenzotriazole, Arch. Biochem. Biophys., 216, 522-529, 1982. 

Oxidation of Phenolic Compounds. Phenolic compounds are widespread throughout the plant kingdom and are prevalent in fruits where they are important contributors to color and flavor (46). Phenolic compounds, particularly flavonoids and derivatives of chlorogenic acid, play a crucial role in the development of a number of postharvest disorders through their oxidation to brown compounds that discolor many fruits and vegetables and substantially reduce their quality. A number of enzymes catalyze the biosynthesis or oxidation of phenolic compounds, among them phenylalanine ammonia lyase (PAL), tyrosine ammonia lyase (TAL), cinnamic acid-4-hydroxylase (CA4H), polyphenol oxidase (PPO), and catechol oxidase (CAO). The chemistry... 

Cinnamate 4-hydroxylases catalyze the hydroxylation of frans-cinnamic acid into trans-p-coumaric acid. The ability to monitor this enzyme activity in Jerusalem artichoke allowed isolation of the P450 enzyme CYP73A1 using conventional chromatography and generation of specific antibodies . ... 

Cinnamic Acid 4-Hvdroxvlase - The p-hydroxylation of cinnamic acid in the biosynthesis of lignins by plants is mediated by a cytochrome P-450 enz3nne.l ° The cinnamic acid 4-hydroxylase of Jerusalem artichoke tubers is preferentially inactivated by 1-aminobenzotriazole in a tlme-and catalysis-dependent manner.The specific inhibition of cytochrome P-450 enzymes in plants provides a possible avenue for the design of new classes of herbicides and other crop-protection agents. 

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.

G. Alibert, R. Ranjeva and A. Boudet, Recherches sur les enzymes catalysant la formation des acides phenoliques chez Quercus ped mculata (Ehrh.). II. Localisation intracellulaire de la phenylalanine ammoniac-lyase, de la cinnamate 4-hydroxylase et de la "benzoate-synthase", Biochim. Biophys. Acta... 

With the failure to demonstrate that norbelladine or its relatives plays a role in the biosynthesis of the mesembrine alkaloids, a reevaluation led to a modified approach in which attempts to identify the sequence of occurrence of the post-tyrosine and post-phenylalanine intermediates were made. There is now a substantial body of information available to suggest that phenylalanine and tyrosine have separate metabolic roles in plants belonging to the order Dictolyoden. Not only do they lack the enzyme phenylalanine hydroxylase (phenylalanine 4-monooxygenase) which is necessary for the conversion of phenylalanine to tyrosine, but the metabolic pathways of these two amino acids are generally quite different in secondary metabolism (70). Phenylalanine is involved in initial conversion to cinnamic acid and subsequent transformation to structural units of the so-called phenyl-propanoid pathway, which include Ar—C3, Ar—C2, and Ar—Cj structural entities. On the other hand, the role of tyrosine in the biosynthesis of secondary metabolities is most frequently seen as the precursor of Ar—Cj—N and Cg—C2—N units, and somewhat less frequently, as Ar—C2 and Q—C2 units. 

The amino acid phenylalanine is derived from gallic acid, being this compound biosynthesized in the shikimic acid metabolic route. Most of the phenolic compounds from higher plants are also derived from this amino acid, formed in the phenylpropanoid metabolic route, in the cell cytoplasm, being various enzymes involved in this metabolism. Phenylalanine ammonia lyase interacts with phenylalanine forming cinnamic acid, that is, hydrolyzed by citmamate-4-hydroxylase, rendering p-coumaric acid. Different hydroxylations and/or methoxylations, of this... 

Resveratrol biosynthesis branches from the phenylpropanoid pathway. The resveratrol biosynthesis pathway consists of four enzymesrphenylalanine ammonia lyase (PAL), cinnamic acid 4-hydroxylase (C4H), 4-coumarate CoA ligase (4CL), and stilbene synthase (STS). The first two enzymes of the pathway, PAL and C4H, convert phenylalanine into /)-coumaric acid. The third enzyme, 4CL, attaches /)-coumaric acid to the pantetheine group of coenzyme-A (CoA) to produce 4-coumaroyl-CoA. The fourth enzyme, STS, catalyzes the condensation of resveratrol from one molecule of 4-coumaroyl-CoA and three molecules of malonyl-CoA, which originate from fatty acid biosynthesis. TAL is homologous to PAL and converts the amino acid tyrosine directly into / -coumaric acid. Methylated resveratrol derivatives of pinostilbene and pterostilbene are produced by resveratrol O-methyltransferase (ROMT) from resveratrol [135] (Figure 10.10). 

Phenylpropanoids are biologically synthesized from phenylalanine as described above. Among them, cinnamic acid is synthesized directly from phenylalanine by phenylalanine ammonia-liase (PAL), and p-hydroxycinnamic acid p-coumaric acid) is synthesized from cinnamic acid by cinnamic acid 4-hydroxylase (C4H, an enzyme in the cytochrome P-450 family).The phenylpropanoid metabolic pathway is important for plants to synthesize lignin, and some phenylpropanoids are seen at junctions of cell wall polysaccharides such as hemicellulose and pectin. 

Phenylalanine hydroxylase occurs only in mammalian liver (that is, in the rat, guinea-pig, rabbit, d<, chicken, and human) (see also 259). No activity has been observed in (rat) lung, kidney, brain, or muscle. The system is quite speciOc for L-phenylalanine. Tjrro-sine is not formed from n-phenylalanine, nor are the corresponding p-phenols formed from N-acetyl- or N-chloroacetyl-L-phenylalanine, L-phenylalanine ethyl ester, DL-phenylglycine, phenylserine, phenylpyruvic acid, phenylethylamine, benzoic acid, hippuric acid, cinnamic acid, or mandelic acid (768), or from aniline, acetanilide, tryptophan, kynurenine, anthranilic acid, or phenylacetate (557). This specificity is a distinguishing character of the enzyme, which occurs in the same tissue as the nonspecific aromatic hydroxylase described above. 

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