Hydroxycinnamoyl-CoA shikimate

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). 

Figure 3-9. Biosynthesis of monolignols. The enzymes involved in this pathway are ( ) hydroxycinnamoyl-CoA shikimate/quinate hydroxy-cinnamoyl transferase, (b) p-coumaroyl-CoA 3 -hydroxylase (E.C. 1.14.14.1), (c) caffeoyl-CoA O-methy 1 Iranslerasc (E.C. 2.1.1.104), (d) cinnamoyl-CoA reductase (E.C. 1.2.1.44) (e) cinnamyl alcohol dehydrogenase (E.C. 1.1.1.195), (f) coniferyl aldehyde/coniferyl alcohol 5-hydroxylase (E.C. 1.14.13), (g) coniferaldehyde/coniferyl alcohol O-methyltransferase (E.C. 2.1.1.68).

Ulbrich, B. and Zenk, M.H. (1980) Partial purification and properties of p-hydroxycinnamoyl-CoA shikimate-p-hydroxycinnamoyl transferase from higher plants. Phytochemistry, 19,1625-9. 

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 ... 

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.

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.

The BAHD superfamily is meanwhile divided into five or eight clades. Although the superfamily is growing fast, there are only three more crystal structures of BAHD enzymes which have been reported. Of them, the latest one is represented by HCT/HQT (hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl-transferase) from Coffea cane-phora. The native HCT has been biochemically and structurally well characterized. Meanwhile only two other structures have been described, an anthocyanin malonyltransferase and trichotecene acetyl-... 

The general phenylpropanoid pathway links the shikimate pathway to the lignin branch pathway. The latter pathway leads to the formation of a series of hydroxycinnamic acids and hydroxycinnamoyl-CoA esters varying in their degrees of hydroxylation and methylation [5]. 

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... 

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. 

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... 

More complex shikimate derivatives have also been produced. One example is the antioxidant, anti-inflammatory, and antiproliferative hydroxycinnamoyl anthranilates (also known as avenanthramides or Avns). The production was first shown from feeding of cinnamates and anthranilates, but the entire pathway from glucose was also demonstrated by using feedback-resistant AroG and TyrA as well as exogenous 4-coumarate CoA ligase from Arabidopsis thcdiana and hydroxycinnamoyl/benzoyl-CoA/anthranilate Af-hydroxycinnamoyl/ benzoyltransferase from Dianthus caryophyllus (carnation) [43]. 

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