4- Coumarate 3-hydroxylase

Figure 5.4. Abbreviated scheme for biosynthesis of major flavonoid subclasses, showing the primary enzymes and substrates leading to different subclasses. Bold-faced, uppercase abbreviations refer to enzyme names, whereas substrate names are presented in lowercase letters. PAL, phenylalanine ammonia lyase C4H, cinnamate 4-hydroxylase 4CL, 4-coumarate CoA ligase CHS, chalcone synthase CHI, chalcone isomerase CHR, chalcone reductase IPS, isoflavone synthase F3H, flavonone 3-hydroxylase F3 H, flavonoid 3 -hydroxylase F3 5 H, flavonoid 3 5 -hydroxylase FNSI/II, flavone synthase DFR, dihydroflavonol 4-reductase FLS, flavonol synthase ANS, anthocyanidin synthase LAR, leucoanthocyanidin reductase ANR, anthocyanidin reductase UFGT, UDP-glucose flavonoid 3-O-glucosyltransferase. R3 = H or OH. R5 = H or OH. Glc = glucose. Please refer to text for more information.

Figure 6.1 Major branch pathways of flavonoid biosynthesis in Arabidopsis. Branch pathways, enzymes, and end products present in other plants but not Arabidopsis are shown in light gray. Abbreviations cinnamate-4-hydroxylase (C4H), chalcone isomerase (CHI), chalcone synthase (CHS), 4-coumarate CoA-ligase (4CL), dihydroflavonol 4-reductase (DFR), flavanone 3-hydroxylase (F3H), flavonoid 3 or 3 5 hydroxylase (F3 H, F3 5 H), leucoanthocyanidin dioxygenase (LDOX), leucoanthocyanidin reductase (LCR), O-methyltransferase (OMT), phenylalanine ammonia-lyase (PAL), rhamnosyl transferase (RT), and UDP flavonoid glucosyl transferase (UFGT).

CL, 4-coumarate CoA ligase CHS, chalcone synthase CHI, chalcone isomerase F3H, flavanone 3-hydroxylase DFR, dihydroflavonol 4-reductase ANS, anthocyanidin synthase FGT, flavonoid 3-O-glucosyltransferase. 

Cinnamate 4-hydroxylase (C4H EC 1.14.13.11, also defined as CYP73A [36]) catalyzes the p-hydroxylation of trani-cinnamate to form trani -p-coumarate. The enzyme has a requirement for molecular oxygen and NADPH as well as association with the electron donor NADPH-cytochrome P450 reductase (CPR EC 1.6.2.4) for activity. C4H was the first characterized plant P450 [37, 38]. 

The PAL activity that is necessary for lignin formation occurs in the cytoplasm or bound to the cytoplasmic surface of the endoplasmic reticulum membranes. The cinnamic acid produced is probably carried on the lipid surface of the membranes, since it is lipophilic, and it is sequentially hydroxylated by the membrane-bound hydroxylases (47,50). In this way there is the possibility of at least a two-step channeling route from phenylalanine to p-coumaric acid. The transmethylases then direct the methyl groups to the meta positions. There is a difference between the transmethylases from angiosperms and those from gymnosperms, since with the latter... 

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. (1). Schematic view of some branches of phenylpropanoid metabolism. Solid arrows indicate enzymatic reactions with the respective enzyme indicated on the right. PAL, phenylalanine ammonia-lyase C4H, cinnamate 4-hydroxylase 4CL, 4-coumarate CoA ligase CHS, chalcone synthase CF1, chalcone flavavone isomerase F3H, flavanone 3-hydroxylase DFR, dihydroflavonol reductase CHR, chalcone reductase. Broken arrows indicate metabolic branches towards several classes of phenylpropanoids, or several subsequent enzymatic steps. In some cases the enzymes indicated are also involved in other reactions, not shown.

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 1.37 Proposed biosynthetic pathway of curcuminoids in tumeric. Enzyme abbreviations CCOMT, caffeoyl-CoA O-methyltransferase 4CL, 4-coumarate CoA ligase CST, shikimate transferase CS3 H, p-coumaroyl 5-O-shikimate 3 -hydroxylase OMT, O-methyltransferase PKS, polyketide synthase. [Adapted from Ramirez-Ahumada et al. (2006)]...

The biosynthetic pathway for salicylic acid is not clear. At present, at least two pathways have been proposed. Each branches from phenyl-propanoid biosynthesis after phenylalanine has been converted to trans-cinnamic acid by phenylalanine ammonium lyase (PAL). In one scheme (Pathway 1 Fig. 4), tram-cinnamic acid would be converted to 2-hydroxy cinnamic acid (or 2-coumaric acid) by a cinnamate 2-hydroxylase. This compound could then be converted to salicylic acid via -oxidation possibly through an acetyl coenzyme A (CoA) intermediate. Alternatively, tram-cinnamic acid could be oxidized to benzoic acid and then hydrox-ylated via a postulated o-hydroxylase activity. The details of this pathway, particularly in tobacco and cucumber, deserve further study. 

Figure 4.1 Current view of the phenylpropanoid metabolism. PAL, phenylalanine ammonia-lyase C4H, cinnamate 4-hydroxylase 4CL, 4-coumarate CoA-ligase HCT, hydroxycinnamoyl-CoAishikimate/quinate hydroxycinnamoyltransferase 3-hydroxylase, 4-hydroxycinnamoylshikimate/quinate 3-hydroxylase.

Phenylalanine ammonia-lyase (PAL) eliminates the amino group from phenylalanine (12) to produce cinnamic acid (13). Cinnamate-4-hydroxylase (C4H) hydroxidizes compound (13) to yield p-coumaric acid (14). 4-CoumaroyhCoA-ligase (4CL) complex catalyzed the conversion of p-coumaric acid (14) and coenzyme A (CoA) to 4-coumaroyl-CoA (15) and 3 moles malonyl-CoA (16). Stilbene synthase (STS) converts these two compounds (15,16) into resveratrol of stilbene (7) (Fig. 3) [23,24],... 

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... 

HAL and PAL Proposed Tyr loop-in model for breathing motion tor substrate access The molecular basis of PAL and TAL substrate versatility Hydroxycinnamoyl CoA Shikimate/Quinate Hydroxycinnamoyltransferase Cytochrome P-450 Hydroxylation Reactions (Cinnamate 4-Hydroxylase, p-Coumarate 3-Hydroxylase , and Ferulate 5-Hydroxylase ) Comparison to Bacterial/Mammalian P-450s Subcellular localization of C4H, pC3H , and F5H ... 

Cinnamate 4-hydroxylase p-Coumarate 3-hydroxylase Ferulate 5-hydroxylase... 

L. B. Davin N. G. Lewis, Reaction tissue formation and stem tensile modulus properties in wild type and p-coumarate-3-hydroxylase downregulated lines of alfalfa, Medicago sativa (Fabaceae), pp 912-925, Copyright 2007, with permission from the Botanical Society of America (g). 

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.

Cytochrome P-450 Hydroxylation Reactions (Cinnamate 4-Hydroxylase, p-Coumarate 3-Hydroxylase , and Ferulate 5-Hydroxylase ) Comparison to Bacterial/ Mammalian P-450s... 

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 . ... 

Fig, 5.2. Biosynthesis of flavonoids and proanthocyanidins (condensed tannins). Enzymes in bold have been cloned from P. tremuloides and show induction by herbivory (Peters and Constabel, 2002 R. Mellway and C. P. Constabel, unpublished data). Abbreviations are as follows Phe, phenylalanine PAL, phenylalanine ammonia lyase 4CL, 4-coumarate CoA Ligase CHS, chalcone synthase CHI, chalcone isomerase F3H, flavanone 3-hydroxylase FLS, flavonol synthase DFR,... 

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