P-coumarate CoA ligase

Biosynthesis of flavonoids starts with the conversion of phenylalanine or tyrosine to cinnamic acid by phenylalanine ammonia lyase (PAL) (Hahlbrock and Grisebach, 1975). Subsequent reactions are catalyzed by cinnamic acid 4-hydrolase to form 4-hydroxyl cinnamic acid (p-coumaric acid). The p-coumaric acid is then catalyzed by p-coumarate CoA ligase to form p-coumaroyl CoA. 

The next major enzyme in the series, p-coumarate CoA ligase, was first isolated from illuminated cell suspension cultures of Petroselinum crispum (syn. P. hortense) and shown to be specifically related to flavonoid and stilbene biosynthesis. The enzyme requires p-coumaric acid, CoASH, ATP, and Mg for activity (Hahlbrock, 1981 Hutchinson, 1986). Three malonyl-CoA units are added and cyclized via a Claisen condensation (see Chapter 5) to produce a chalcone intermediate (9). Condensation of the same intermediates via an aldol condensation yields pyrones and... 

Cinnamate Co A ligase, p-coumarate CoA ligase 2 cinnamoyl CoA reductase 3 cinnamoyl alcohol dehydrogenase... 

Figure 1.15 Schematic of the main pathways and key enzymes involved in the biosynthesis of hydro-lysable tannins, salicylic acid, hydroxycinnamates and 5-caffeoylquinic acid. Enzyme abbreviations PAL, phenylalanine ammonia-lyase BA2H, benzoic acid 2-hydroxylase C4H, cinnamate 4-hydroxylase COMT-1, caffeic/5-hydroxyfemlic acid O-methyltransferase 4CL, p-coumarate CoA ligase F5H, ferulate 5-hydroxylase CT, galloyltransferase ACoAC, acelylCoA carboxylase.

An alternate fate of the products of photosynthesis that are channeled through the shikimate pathway is for 3-dehydroshikitnic acid to be directed to L-phenylalanine and so enter the phenylpropanoid pathway (Figure 1.15). Phenylalanine ammonia-lyase catalyses the first step in this pathway, the conversion of L-phenylalanine to cinnamic acid, which in a reaction catalysed by cinnamate 4-hydroxylase is converted to p-coumaric acid which in turn is metabolized to p-coumaroyl-CoA by p-coumarate CoA ligase. Cinnamic add is... 

Fig. 12. Tentative model of the signal transduction chain that links the perception of pectic fragments to defense responses in carrot cells. Abbreviations apy, heterotrimeric G protein CaM, calmodulin 4CL, 4-coumarate-CoA ligase CTX, cholera toxin FC, fusicoccine GDP-P-S and GTP-y-S, guanosine 5 -0-(2-thiodiphosphate) and guanosine 5 -0-(3-thiotriphosphate) IP3, 1,4,5-inositol trisphosphate PAL, phenylalanine ammonia-lyase PLC, phospholipase C PR, pathogenesis related PTX, pertussis toxin Rc, receptor SP, staurosporine. Activation and inhibition are symbolized by + and -respectively.

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)]...

Schneider, K., Hoevel, K., Witzel, K., Hamberger, B., Schomburg, D., Kombrink, E. and Stuible, H.P. (2003) The substrate specificity-determining amino acid code of 4-coumarate CoA ligase. Proc. Natl. Acad. Sci. USA., 100,8601-06. 

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,... 

The first step of flavonone biosynthesis begins with the deamination of the amino acid phenylalanine or tyrosine by a phenylalanine ammonia-lyase (PAL) or a tyrosine ammonia-lyase (TAL), which affords cinnamic acid and p-coumaric acid, respectively (Figure 6.36). The formed cinnamic acid is first hydroxylated to p-coumaric acid by a membrane-bound P450 monooxygenase, cinnamate 4-hydroxylase (C4H), and then activated to p-coumaroyl-CoA by a 4-coumarate-CoA ligase (4CL). 4CL catalyzes also the conversion of caffeic acid, feruhc acid, and cinnamic acid to caffeoyl-CoA, feruloyl-CoA, and cinnamoyl-CoA, respectively. 

Cinnamic (1), p-coumaric (2), and related acids may be activated by conversion to CoA esters by CoA ligases [e.g., 4-coumarate CoA ligase (EC 6.2.1.12)] in much the same way that fatty acids are activated. The reduction of the CoA esters of cinnamic acids to cinnamyl alcohols involves two enz)mies cinnamoyl-CoA oxidoreductase (which forms the aldehydes) and cinnamyl alcohol dehydrogenase (Grisebach, 1981). Phenylpropanoids appear to be synthesized from the CoA esters of this series of acids by conversion to the corresponding aldehydes, then to the alcohols, and finally, by elimination of a phosphate group, to allyl and propenyl compounds. In many plants, mixtures of all t3q>es co-occur (Fig. 8.7) (Gross, 1981 Mann, 1987). Reduction of the side chain to produce dihydrocinnamic acids and related compounds is also known to occur in nature. 

Coumarin biosynthesis involves formation of PAL and 4-coumarate CoA ligase which is light and elicitor inducible. Cells of P. crispum respond by producing the linear furanocoumarins psoralen (17), bergapten (18), and xanthotoxin (19) (Fig. 9.6), as well as other furanocoumarins (Ellis, 1988). The relative amounts of furanocoumarins in the mixture was changed by use of different fungal elicitor sources. 

Finally, in this vein, as shown in Scheme 12.23, p-coumaric acid is converted (in the presence of 4-coumarate-CoA ligase, EC 6.2.1.12, coenzyme A, and ATP) into its corresponding coenzyme A thioester, 4-coumaroyl-CoA. Then, in the presence of the enzyme naringenin chalcone synthase (EC 2.3.1.74), 3 equivalents of malonyl-CoA (Scheme 11.36) are added sequentially, a cycUzation follows, and the chalcone (naringenin chalcone) is produced. This is the beginning of the pathway for flavo-noid (as derived from this progenitor chalcone) biosynthesis. 

The experiment described in the beginning of this section involved the incorporation of four heterologous genes S. cerevisiae PAL, Streptomyces coelicolor A3 cinnamate/coumarate CoA ligase (ScCCL) with substrate specificity toward both cinnamic acid 5 and p-coumaric acid 6, licorice plant Glycyrrhiza echinatd) CHS,... 

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],... 

The biosynthetic pathway for isoflavonoids in soybean and the relationship of the isoflavonoids to several other classes of phenylpropanoids is presented in Fig. 8.2. Production of /i-coumaryl-CoA from phenylalanine requires phenylalanine ammonia lyase to convert phenylalanine to cinnamate, cinnamic acid hydroxylase to convert cinnamate to /7-coumarate, and coumaraterCoA ligase to convert jt -coumarate to -coumaroyl-CoA. Lignins may be produced from j3-coumaroyl-CoA or from />-coumarate. Chalcone synthase catalyzes the condensation of three molecules of malonyl CoA with p-coumaroyl-CoA to form 4, 2 , 4 , 6 -tetrahydroxychalcone, which is subsequently isomerized in a reaction catalyzed by chalcone isomerase to naringenin, the precursor to genistein, flavones, flavonols, condensed tannins, anthocyanins, and others. 

Caffeate CoA ligase (D 22.2.1) p-Coumaric acid and caffeic acid esters of quinic acid I... 

Methylation of caffeic acid leads to the formation of femlic acid, which, together with p-coumaric acid, are the precursors of lignins. The methylation is catalyzed by an 0-methyltransferase. Caffeic acid is the substrate for the rare 5-hydroxyfemlic acid, which yields sinapic acid as a result of O-methylation. The formation of hydroxyciimamic acid derivatives requires the formation of hydroxycinnamate-CoAs (e.g., p-coumaroyl-CoA) catalyzed by hydroxyciimamoyl-CoA ligases or by the action of 0-glycosyl transferases. 

Figure 1. Proposed biosynthetic pathway to monolignols, and related metabolites, a. Phenylalanine ammonia-lyase, b. Tyrosine ammonia-lyase, c. Cinnamate-4-hydroxylase, d. p-Coumarate-3-hydroxylase, e. O-Methyltransferase, f. Ferulate-5-hydroxylase, g. Hydroxycinnamate-CoA ligase, h. Cinnamoyl-CoA NADP oxidoreductase, i. Cinnamyl alcohol dehydrogenase. ( Note plus acetate pathway for both flavonoids and suberin)...

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