Caffeoyl CoA

CHS carries out a series of sequential decarboxylation and condensation reactions, using 4-courmaroyl-CoA (in most species) and three molecules of malonyl-CoA, to produce a poly-ketide intermediate that then undergoes cyclization and aromatization reactions that form the A-ring and the resultant chalcone structure. The chalcone formed from 4-courmaroyl-CoA is naringenin chalcone. However, enzyme preparations and recombinant CHS proteins from some species have been shown to accept other HCA-CoA esters as substrates, such as cinnamoyl-CoA (see, e.g., Ref. 37). In particular, the Hordeum vulgare (barley) CHS2 cDNA encodes a CHS protein that converts feruloyl-CoA and caffeoyl-CoA at the highest rate, and cinnamoyl-CoA and 4-courmaroyl-CoA at lower rates. 

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.

The substitution of the phenyl ring necessary for the biosynthesis of coniferyl alcohol (3.79) and sinapyl alcohol (3.81) begins with the hydroxylation of C3. This is a conversion that requires the formation of the ester of /5-coumaroyl-CoA with D-quinate (3.73) or shikimate (3.74) catalyzed by the enzyme hydroxycinnamoyl-CoA shikimate/quinate hydroxy-cinnamoyl transferase (HCT Hoffmann et al., 2003). The hydroxylation of this ester intermediate is catalyzed by the enzyme /i-coumarovl-Co A 3 -hydroxylase (C3 H Schoch et al., 2001 Franke et al., 2002a,b). The resulting shikimate or quinate ester (3.75 3.76) is subsequently hydrolyzed by the same HCT, resulting in caffeoyl-CoA (3.36). 

Kuhnl, T., Koch, U., Heller, W., and Wellmann, E., 1989, Elicitor induced. S -adcnosyl-L-methionine caffeoyl-CoA 3 YYmcthvl transferase from carrot cell suspension, Plant Sci. 60 21-25. 

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)]...

A calcium atom is observed in the active site, surrounded by an octahedral coordination shell. The side chain oxygens of Thr 63, Glu 67, Asp 163, Asp 189, and Asn 190 are involved in the chelation of this calcium atom, completed by the 3-hydroxyl group of caffeoyl CoA or 5-hydroxyferuloyl CoA and a water molecule. This calcium atom substitutes the Mg2+ atom expected at this position, and is present in the catechol OMT structure with the same coordination geometry., 38 In CCoAOMT, Mg2+ mediates the deprotonation of the caffeoyl 3-hydroxyl group and maintains the 3-hydroxyl group in close proximity to the reactive methyl group of SAM ( 3A), suitably positioned for facile transmethylation to occur (Fig. 2.8). 

Guo, D., Chen, R, Inoue, K., Blount, J.W. and Dixon, R.A. (2001) Downregulation of caffeic acid 3-O-methyltransferase and caffeoyl CoA 3-O-methyltransferase in transgenic alfalfa Impacts on lignin structure and implications for the biosynthesis of G and S lignin. Plant Cell, 13, 73-88. 

Schmitt, D., Pakusch, A.E. and Matem, U. (1991) Molecular cloning, induction and taxonomic distribution of caffeoyl-CoA 3-O-methyltransferase, an enzyme involved in disease resistance.. Biol. Chem., 266,17416-23. 

Hydroxycinnamoyl CoAishikimate/quinate hydroxycinnamoyltransferase (HCT, EC 2.3.1.133) catalyzes esterification of shikimate (29) and quinate (28) with -coumaroyl CoA (9) to afford the corresponding esters 25 and 24, respectively. This biochemical conversion was discovered by Stockigt and Zenk, with later studies describing its partial purification and the reversibility of the enzymatic conversion. Interestingly, this step has now been established as the forerunner to the second hydroxylation (at C3) to ultimately afford caffeoyl CoA (10) (see Figure 4). As for PAL/TAL, this protein is considered cytosolic, as the enzyme contains... 

Cloning of a cDNA encoding HCT from tobacco N. tabacum, NtHCT) stems and expression of the recombinant protein in fully functional form in E. coli as a fusion product have also been carried out, with kinetic parameters of the purified protein determined (C. L. Cardenas etal, manuscript in preparation). " The overall catalytic propenies of recombinant NtHCT with various hydroxycinnamoyl CoAs (9-13) using either shikimic (29) or quinic (28) acid as substrate were quite informative (C. L. Cardenas et al, manuscript in preparation). With shikimic acid (29) at a saturating concentration, the observed /feat/Am of recombinant NtHCT established that the dominant HCT activity was with -coumaroyl CoA (9) (114840mor ls ) over that of caffeoyl (10), feruloyl (11), or sinapoyl (13) CoA (48 420, 6880, and 420 moF 1 s , respectively). Recombinant NtHCT was able, however, to less efficiently transfer hydroxycinnamoyl moieties from -coumaroyl CoA (9) and caffeoyl CoA (10) to quinic acid (28). Indeed, /fcat/Am values at saturating concentration of quinic acid (28) were lower by approximately 7.5- and 131-fold for/)-coumaroyl CoA... 

SlOmoF ls ) and caffeoyl CoA (10) (370moF ls ), respectively, on comparison to catalytic turnover of NtHCT with shikimic acid (29) (saturating substrate). 

The kinetic studies of the recombinant NtHCT also further provided evidence for the reverse reaction towards formation of the hydroxycinnamoyl CoAs 9 and 10 (C. L. Cardenas etal, manuscript in preparation). In the presence of coenzyme A, NtHCT catalyzed cleavage of the ester bond of the respective hydroxycinnamoyl shikimate esters 25 and 27. The reverse reaction kinetics demonstrated that NtHCT was able to convert -coumaroyl shikimate (25) to -coumaroyl CoA (9) and caffeoyl shikimate (27) to caffeoyl CoA (10), with cat/Am values of 31 000 and 19 llOmoF Is , respectively, at saturating concentrations of CoA. 

As indicated in both earlier and more recent critical reviews, there was significant confusion as regards the enzymes involved in 0-methylation in the monolignol pathway and the substrates utilized. However, it is now known that two classes of cytosolic 0-methyltransferases (OMTs) are involved caffeic acid 0-methyltransferase (COMT, EC 2.1.1.68) and caffeoyl CoA 0-methyltransferase (CCOMT, EC 2.1.1.104) (see Figure 4). 

The preceding (or first monolignol pathway) methylation step was ultimately discovered to utilize a CoA derivative, that is, caffeoyl CoA (10), to afford the corresponding feruloyl derivative (11), rather than the corresponding cafifeic acid (5) to produce ferulic acid (6). Taken together, these studies had established that two quite distinct biochemical processes were operative for each methylation step in monolignol biosynthesis. 

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