The monolignols are the building blocks of lignans (Section 11) and lignin (Section 12), whereas some of the intermediates of monolignol biosynthesis serve as precursors for hydroxycinnamic acids (Section 13) and sinapoyl esters (Section 14). Monolignols are synthesized from p-coumaroyl-CoA (3.31) generated via the shikimate and general phenylpropanoid pathways (see Sections 6 and 7). As part of monolignol biosynthesis p-coumaroyl-CoA (3.31) can undergo two types of modifications: reduction of the carboxyl group on the propane side chain to an alcohol, and substitution of the phenyl ring (Figure 3-9). The two predominant monolignols are coniferyl alcohol (3.79) and sinapyl alcohol (3.81). p-Coumaryl alcohol (3.70) and 5-hydroxyconiferyl alcohol (3.80) are generally much less abundant, and are found only in trace amounts in some species or tissues.
The reduction of p-coumaroyl-CoA (3.31) to p-coumaryl aldehyde (3.69) is catalyzed by the enzyme cinnamoyl-CoA : NADP oxidoreductase (CCR). This enzyme was initially purified from soybean cultures (Wegenmayer et al., 1976), and was later on efficiently isolated from lignifying cambium of eucalyps (Eucalyptus gunnii) (Goffner et al., 1994). A CCR cDNA was identified in a cDNA library that was screened with oligonucleotiede derived from the peptide sequence of the CCR protein. CCR is considered the first enzyme committed towards the biosynthesis of monolignols and shows homology to the flavonoid biosynthetic gene flavonol 4-reductase (Lacombe et al., 1997).
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 p-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 p-coumaroyl-CoA 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).
The enzyme responsible for the hydroxylation of C3 was extremely difficult to identify. It had been postulated to be a phenol oxidase, dioxygenase, or a cytochrome P450 monooxygenase, but biochemical approaches aimed at isolating a protein displaying activity toward p-coumaric acid (3.30) were unsuccessful. With the availability of the Arabidopsis thaliana genome sequence, Schoch et al. (2001) performed a phylogenetic analysis of the genes encoding cytochrome P450 enzymes (for a review on this class of enzymes, see Chapple (1998)). This analysis resulted in the identification of CYP98A3 as a putative C3H. Expression analyses confirmed that the gene encoding CYP98A3 was expressed in tissues that would be expected to synthesize caffeic acid derivatives, including lignified tissues, and wounded tissues that produced chlorogenic acid (1.18). After cloning of the corresponding cDNA and expression of the recombinant enzyme in yeast, substrate specificity could be investigated. The enzyme did not show activity towards p-coumaric acid (3.30), p-coumaroyl-CoA (3.31), the p-coumaroyl glucose ester, nor the p-coumaroyl 4-glucoside. Based on experiments by Heller and Kuhnl (1985) and Kuhnl et al. (1987) with parsley cell suspension cultures, Schoch et al. (2001) were able to show activity of the recombinant CYP98A3 enzyme towards the shikimate and D-quinate esters of p-coumaroyl-CoA (3.71; 3.72), which resulted in the shikimate and D-quinate esters of p-caffeoyl CoA (3.73; 3.74), respectively. When the numbering of carbon atoms in the p-coumaroyl-CoA esters is taken into consideration, the carbon at the 3' position - not the 3 position - is hydroxylated. Consequently, the enzyme is now referred to as C3'H. Franke et al. (2002a) showed that the Arabidopisis reduced epidermal fluorescence8 (ref8) mutant was unable to synthesize caffeic acid (3.32) as a result of a defective copy of the C3H gene.
Figure 3-9. Biosynthesis of monolignols. The enzymes involved in this pathway are: (a) hydroxycinnamoyl-CoA shikimate/quinate hydroxy-cinnamoyl transferase, (b) p-coumaroyl-CoA 3'-hydroxylase (E.C. 126.96.36.199), (c) caffeoyl-CoA O-methyltransferase (E.C. 188.8.131.52), (d) cinnamoyl-CoA reductase (E.C. 184.108.40.206) (e) cinnamyl alcohol dehydrogenase (E.C. 220.127.116.11), (f) coniferyl aldehyde/coniferyl alcohol 5-hydroxylase (E.C. 1.14.13), (g) coniferaldehyde/coniferyl alcohol O-methyltransferase (E.C. 18.104.22.168).
HCT, the enzyme responsible for the formation of the D-quinate and shikimate esters of p-coumaroyl CoA (3.71; 3.72), was identified by Hoffmann et al. (2003) in stem extracts of tobacco. Separation of the proteins with HPLC resulted in a fraction containing HCT activity. The protein was partially sequenced, and degenerate primers were synthesized to amplify the corresponding cDNA. Purification of the recombinant protein from E. coli enabled more detailed studies on substrate specificity and catalytic properties. These studies showed that HCT was able to catalyze the esterification reaction of D-quinate and shikimate with both p-coumaroyl-CoA (3.31) and caffeoyl-CoA (3.36). The enzyme could also catalyze the reverse reaction, i.e. the hydrolysis of the ester, thus producing caffeoyl CoA and either D-quinate or shikimate. The role of this enzyme in phenylpropanoid metabolism was further demonstrated via down-regulation of the HCT gene in Arabidopsis and tobacco (Nicotiana benthamiana). The transgenic plants were dwarfed, accumulated caffeoylquinate esters in their leaves, and showed different lignin subunit composition (Hoffmann et al., 2004).
Caffeoyl-CoA (3.36) is methylated to feruoyl-CoA (3.75) by the enzyme caffeoyl-CoA O-methyltransferase (CCoA-OMT). CCoA-OMT had been implicated in disease responses based on its induction in carrot (Daucus carota) cell suspension cultures that were treated with elicitors (Kuhnl et al., 1989). A more general role of CCoA-OMT in phenylpropanoid metabolism was proposed after Ye et al. (1994) showed that the CCoA-OMT gene was up-regulated during the in vitro development of lignified tracheary elements derived from Zinnia elegans mesophyl cells. Feruoyl-CoA (3.75) is subsequently reduced to coniferaldehyde (3.76) by CCR, analogous to the reduction of p-coumaroyl CoA (3.31) to p-coumaryl aldehyde (3.69).
Coniferaldehyde (3.76) can undergo several fates, some of which can ultimately lead to the same end product. It can be reduced to coniferyl alcohol (3.79) by the enzyme cinnamyl alcohol dehydrogenase (CAD). Alternatively, the enzyme coniferyl aldehyde/coniferyl alcohol 5-hydroxylase (C5H), also known by its less accurate name ferulic acid 5-hydroxylase (F5H; Humphreys et al., 1999) can catalyze the hydroxylation of C5 to result in 5-hydroxy coniferyl aldehyde (3.77). C5H is also able to form 5-hydroxyconiferyl alcohol (3.80) from coniferyl alcohol (3.79). This enzyme was initially identified as F5H, after analysis of the Arabidopsis ferulic acid hydroxylase 1 (fah1) mutant, which was isolated in a mutant screen based on reduced levels of the UV-fluorescent sinapoyl esters (Section 13; Chapple et al., 1992). The FAH1 gene was cloned using a T-DNA tagged mutant allele (Meyer et al., 1996), which revealed that the gene encoded a cytochrome P450 monooxygenase with homology to flavonoid 3', 5' hydroxylases. Substrate specificity of recombinant F5H was evaluated by Humphreys et al. (1999) and Osakabe et al. (1999). Their analyses revealed that F5H had much higher activity towards coniferaldehyde (3.76) and coniferyl alcohol (3.79) than against ferulic acid (3.33).
Methylation of 5-hydroxyconiferyl aldehyde (3.77) and 5-hydroxy coniferyl alcohol (3.80) by the enzyme 5-hydroxyconiferaldehyde/ 5-hydroxyconiferyl alcohol O-methyltransferase results in sinapaldehyde (3.78) and sinapyl alcohol (3.81), respectively. The enzyme catalyzing this step is known by the historic but inaccurate name caffeic acid O-methyl transferase (COMT). So COMT is now thought to be responsible for the methylation of the hydroxyl group on C5, whereas CCoA-OMT is responsible for methylation of the hydroxyl group on C3. This explains why mutations in the COMT gene, such as in the maize brown midrib3 mutant (Vignols et al., 1995) and the sorghum brown midrib26 mutant (Bout and Vermerris, 2003), result in reductions in lignin units derived from sinapyl alcohol, and not in lignin subunits derived from coniferyl alcohol (see also Section 12).
As described above, sinapyl alcohol (3.81) can be synthezed via methylation of 5-hydroxyconiferyl alcohol (3.80) by COMT. An alternative route is via the reduction of sinapaldehyde (3.78) by CAD or, in the case of aspen (Populus tremuloides) and several other angiosperm trees, sinapyl alcohol dehydrogenase (SAD; Li et al, 2001). SAD cDNA's were identified as a distinct class of hybridizing fragments during the screening of an aspen cDNA library derived from lignifying xylem tissue with a probe derived from an aspen CAD cDNA. Analysis of the substrate specificity of the recombinant protein generated by expression of a SAD cDNA in E.coli indicated that SAD had a 60-fold higher affinity for sinapaldehyde than coniferaldehyde.
CAD is encoded by a multigene family in Arabidopsis (Raes et al., 2003; Goujon et al., 2003) and rice (Oryza sativa; Tobias and Chow, 2005), and probably in many other species. Mutants of Arabidopsis in which the genes encoding two distinct isoforms of CAD, CAD-C and CAD-D, were down-regulated as a result of T-DNA insertions were analyzed by Sibout et al. (2003). The reduction in CAD-C activity resulted in minor changes in lignin composition, whereas reduction CAD-D activity resulted in a 45% and 24% reduction in lignin residues derived from sinapyl alcohol in stem and root tissue, respectively. Taken together with the fact that both isoforms display activity towards both coniferaldehyde and sinapaldehyde, these data suggest that the CAD enzymes in Arabidopsis do not display the same substrate specificity for either sinapaldehyde or coniferaldehyde as was observed in aspen. Rather, in Arabidopsis, and possibly many other species, the biosynthesis of coniferyl alcohol and sinapyl alcohol appears to be catalyzed by a combination of isoforms, some of which have a preference towards one of the substrates. The combination of isoforms varies depending on the developmental stage and the tissue (Sibout et al., 2003).
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