Phenylpropanoid pathway enzymes

The key reaction that links primary and secondary metabolism is provided by the enzyme phenylalanine ammonia lyase (PAL) which catalyzes the deamination of l-phenylalanine to form iran.v-cinnamic acid with the release of NH3 (see Fig. 3.3). Tyrosine is similarly deaminated by tyrosine ammonia lyase (TAL) to produce 4-hydroxycinnamic acid and NH3. The released NH3 is probably fixed by the glutamine synthetase reaction. These deaminations initiate the main phenylpropanoid pathway. 

The 4-coumarate CoA ligase (4CL EC 6.2.1.12) enzyme activates 4-coumaric acid, caffeic acid, ferrulic acid, and (in some cases) sinapic acid by the formation of CoA esters that serve as branch-point metabolites between the phenylpropanoid pathway and the synthesis of secondary metabolites [46, 47]. The reaction has an absolute requirement for Mg " and ATP as cofactors. Multiple isozymes are present in all plants where it has been studied, some of which have variable substrate specificities consistent with a potential role in controlling accumulation of secondary metabolite end-products. Examination of a navel orange EST database (CitEST) for flavonoid biosynthetic genes resulted in the identification of 10 tentative consensus sequences that potentially represent a multi-enzyme family [29]. Eurther biochemical characterization will be necessary to establish whether these genes have 4CL activity and, if so, whether preferential substrate usage is observed. 

Myristicin content of some carrot samples was increased two-to five-fold over nonirradiated controls (Table I). The increase in concentration of myristicin is presumed to be via the phenylpropanoid pathway phenylalanine ammonia-lyase, an enzyme of that system, is activated by light (9). Failure of some samples exposed to UV light to synthesize myristicin may be due to the absence or inhibition of a key enzyme needed for myristicin synthesis. 

The tightly regulated pathway specifying aromatic amino acid biosynthesis within the plastid compartment implies maintenance of an amino acid pool to mediate regulation. Thus, we have concluded that loss to the cytoplasm of aromatic amino acids synthesized in the chloroplast compartment is unlikely (13). Yet a source of aromatic amino acids is needed in the cytosol to support protein synthesis. Furthermore, since the enzyme systems of the general phenylpropanoid pathway and its specialized branches of secondary metabolism are located in the cytosol (17), aromatic amino acids (especially L-phenylalanine) are also required in the cytosol as initial substrates for secondary metabolism. The simplest possibility would be that a second, complete pathway of aromatic amino acid biosynthesis exists in the cytosol. Ample precedent has been established for duplicate, major biochemical pathways (glycolysis and oxidative pentose phosphate cycle) of higher plants that are separated from one another in the plastid and cytosolic compartments (18). Evidence to support the hypothesis for a cytosolic pathway (1,13) and the various approaches underway to prove or disprove the dual-pathway hypothesis are summarized in this paper. 

To what extent is the response of cytosolic and plastidic isozymes of the shikimate pathway coordinated or coupled with one another and to alterations in expression of enzymes of the flavonoid and phenylpropanoid-pathway segments Some of the emerging information is given in Figure 6. Thus, light induction, well known to induce PAL and enzymes of the flavonoid pathway, also induces both DS-Mn and DS-Co in parsley cell cultures (49). However, only the cytosolic CM-2 (and not the plastidic CM-1) was induced. Fungal elicitor was reported to induce only DS-Mn—not DS-Co or either of the chorismate mutase isozymes (49). Previous studies... 

In a recent study (54), we showed increased activities of two enzymes of the general phenylpropanoid pathway, PAL and 4-coumarate CoA lig-ase, as well as one enzyme of the specific pathway of lignin biosynthesis, cinnamy 1-alcohol dehydrogenase (CAD), in resistant plants at the time of the hypersensitive host cell death. On the other hand, decreased activities were observed at the same time with susceptible host plants (54). Furthermore, we showed that the well known increase in peroxidase activities, which is strong in resistant and only weak in susceptible plants (55-58), is at least partly due to the increased activity of the lignin biosynthetic pathway (54,59). 

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. ... 

Figure 3.8. The phenylpropanoid pathway, leading to Cg-Cj-Cs phenylpropanoids. Note the versatile use of some individual enzymes in the phase 2 tailoring. From Aksamit-Stachurska A, Korohczak-Sosna A, Kulma A, Szopa J. (2008). Glycosyltransferase efficiently controls phenylpropanoid pathway. BMC Biotechnology, 8, 25-41.

The general phenylpropanoid pathway begins with the deamination of L-phenylalanine to cinnamic acid catalyzed by phenylalanine ammonia lyase (PAL), Fig. (1), the branch-point enzyme between primary (shikimate pathway) and secondary (phenylpropanoid) metabolism [5-7]. Due to the position of PAL at the entry point of phenylpropanoid metabolism, this enzyme has the potential to play a regulatory role in phenolic-compound production. The importance of this is illustrated by the high degree of regulation both during development as well as in response to environmental stimuli. 

Studies have shown that phenylpropanoid metabolism can be stimulated by ozone. The activity of PAL increased in soybean [91], Scots pine (Pinus sylvestris L.) [92], and parsley (Petroselinum crispum L.) [93] soon after treatment with 150-200 nmol O3 mol 1. Rapid increases in transcript levels for PAL in response to ozone have been observed in parsley [93], Arabidopsis thaliana L. Heynhold [94] and tobacco (Nicoticma tabacum L.) [95]. Transcript levels for 4-coumarate CoA ligase (4CL), the last enzyme in the general phenylpropanoid pathway, increased commensurately with PAL transcripts in ozone-treated parsley seedlings [93]. Phenolic compunds reported to accumulate in leaf tissue in response to ozone include hydroxycinnamic acids, salicylic acid, stilbenes, flavonoids, furanocoumarins, acetophenones, and proanthocyanidins [85, 92, 93, 96, 97]. 

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.

One of the major features of phenylpropanoid metabolism is the diversity of end products. The set of enzymic reactions leading from phenylalanine to 4-coumaroyl coenzyme A is common to pathways which lead to these diverse end products and is known as the general phenylpropanoid pathway (Fig. 1). Those biochemical reactions which lead to the synthesis of specialised products are known as branch pathways. 

Table 1. Genes encoding enzymes of the general phenylpropanoid pathway...

In this chapter I will focus on biochemical and molecular aspects leading to lignin production. We have studied in detail phenylalanine ammonia lyase (PAL EC 4.3.1.5), the first enzyme of the general phenylpropanoid pathway, and cinnamyl alcohol dehydrogenase (CAD EC 1.1.1.195), an enzyme specific to the branch pathway leading to lignin formation. 

One aspect shared with several other genes of the phenylpropanoid pathway is the transient induction after environmental challenge. This has also been demonstrated for chalcone synthase (Ryder et al., 1984) and chalcone isomerase (Cramer et al., 1985 Mehdy Lamb, 1987), enzymes involved in phytoalexin production, and for cinnamyl alcohol dehydrogenase (CAD) an enzyme of lignin biosynthesis, in response to elicitor treatment of bean tissue culture cells (Grand et al., 1987). 

Fig. 1. Simplified diagram of the phenylpropanoid and flavonoid biosynthetic pathways. Enzymes that catalyze the reactions are placed on the left-hand side, and transcription factors on the right-hand side of the arrows. Both transcription factors for which their control over the enzymatic steps has been genetically proven, as well as transcription factors that have been shown to interact with promoters of the structural genes, are shown. PAL Phenylalanine ammonia lyase C4H cinnamate 4-hydroxylase 4CL 4-coumaroyl-coenzyme A ligase CHS chalcone synthase CHI chalcone-flavanone isomerase F3H flavanone 3(3-hydroxylase DFR dihydroflavonol 4-reductase AS anthocyanin synthase UFGT UDP glucose-flavonol glucosyl transferase RT anthocyanin rhamnosyl transferase...

Recently, curcuminoid synthase has been identified as being capable of forming the curcuminoids in turmeric (Maria et al., 2006). This activity required malonyl-CoA and phenylpropanoid pathway-derived hydroxycinnamoyl-CoA esters as substrates, suggesting that the corresponding protein was a polyketide synthase or an enzyme that was closely related. It is postulated that this activity could be the result of a single enzyme, or of multiple enzymes in sequence. 

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