Phenylpropanoid biosynthesis

The earliest references to cinnamic acid, cinnamaldehyde, and cinnamyl alcohol are associated with thek isolation and identification as odor-producing constituents in a variety of botanical extracts. It is now generally accepted that the aromatic amino acid L-phenylalanine [63-91-2] a primary end product of the Shikimic Acid Pathway, is the precursor for the biosynthesis of these phenylpropanoids in higher plants (1,2). 

Walker, K., Fujisaki, S., Long, R. and Croteau, R. (2002) Molecular cloning and heterologous expression of the C-13 phenylpropanoid side chain-CoA acyltransferase that functions in Taxol biosynthesis. Proceedings of the National Academy of Sciences of the United States of America, 99, 12715-12720. 

Plant metabolism can be separated into primary pathways that are found in all cells and deal with manipulating a uniform group of basic compounds, and secondary pathways that occur in specialized cells and produce a wide variety of unique compounds. The primary pathways deal with the metabolism of carbohydrates, lipids, proteins, and nucleic acids and act through the many-step reactions of glycolysis, the tricarboxylic acid cycle, the pentose phosphate shunt, and lipid, protein, and nucleic acid biosynthesis. In contrast, the secondary metabolites (e.g., terpenes, alkaloids, phenylpropanoids, lignin, flavonoids, coumarins, and related compounds) are produced by the shikimic, malonic, and mevalonic acid pathways, and the methylerythritol phosphate pathway (Fig. 3.1). This chapter concentrates on the synthesis and metabolism of phenolic compounds and on how the activities of these pathways and the compounds produced affect product quality. 

Ring B and the central three-carbon bridge forming the C ring (see Fig. 5.1) originate from the amino acid phenylalanine, itself a product of the shikimate pathway, a plastid-based process which generates aromatic amino acids from simple carbohydrate building blocks. Phenylalanine, and to a lesser extent tyrosine, are then fed into flavonoid biosynthesis via phenylpropanoid (C6-C3) metabolism (see Fig. 5.1). 

WISMAN, E., HARTMANN, U., SAGASSER, M., BAUMANN, E., PALME, K., HAHLBROCK, K., SAEDLER, H., WEISSHAAR, B., Knock-out mutants from an En-1 mutagenized Arabidopsis thaliana population generate phenylpropanoid biosynthesis phenotypes, Proc. Natl. Acad. Sci. USA, 1998,95,12432-12437. 

BOREVITZ, J.O., XIA, Y, BLOUNT, J DIXON, R.A., LAMB, C., Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis, Plant Cell,... 

FRICK, S., KUTCHAN, T.M., Molecular cloning and functional expression of O-methyltransferases common to isoquinoline alkaloid and phenylpropanoid biosynthesis, Plant J., 1999,17, 329-339. 

Validation of the role of femloyl-CoA in the synthesis of the vanillin precursor will be detection of the appropriate intermediates and/or enzyme activities in placental extracts that could account for the production of the predicted levels of capsaicinoids. The presence of low levels of monolignol intermediates could be explained by lignin biosynthesis. An alternate route from phenylalanine to vanillin has been considered by some investigators Orlova et al. [68] demonstrated the role of the benzenoid pathway in petunia flowers for the biosynthesis of phenylpropanoid/benzenoid volatiles. 

The functions of phenylpropanoid derivatives are as diverse as their structural variations. Phenylpropanoids serve as phytoalexins, UV protectants, insect repellents, flower pigments, and signal molecules for plant-microbe interactions. They also function as polymeric constituents of support and surface structures such as lignins and suberins [1]. Therefore, biosynthesis of phenylpropanoids has received much interest in relation to these functions. In addition, the biosynthesis of these compounds has been intensively studied because they are often chiral, and naturally occurring samples of these compounds are usually optically active. Elucidation of these enantioselective mechanisms may contribute to the development of novel biomimetic systems for enantioselective organic synthesis. 

Neolignans are also compounds composed of two phenylpropanoid units, but linked in a manner other than C8-C8 (Fig. 12.1) [10]. The compounds of this class are often chiral and naturally occurring neolignans are usually optically active, which evokes the involvement of DPs in the biosynthesis of neolignans. 

The mechanism was confirmed by enzymatic experiments [20, 21]. A crude enzyme preparation from A. officinalis cultured cells catalyzed the conversion of j9-coumaryl alcohol and j9-coumaroyl CoA to (Z)-hinokiresinol [20], while a crude enzyme preparation from Cryptomeria japonica cultured cells mediated the formation of ( )-hinokiresinol from the same substrates [21]. In addition, both enzyme preparations converted j9-coumaryl j9-coumarate into (Z)-hinokiresinol and ( )-hinokiresinol [20, 21]. Thus, the biosynthesis of hinokiresinol originating from phenylpropanoid monomers was established. 

Using this system, (Z)-hinokiresinol isolated from cultured cells of A. officinalis was determined to be the optically pure (75 )-isomer, while ( )-hinokiresinol isolated from cultured cells of C. japonica had 83.3% e.e. in favor of the (7S)-enantiomer (Table 12.1). The enzymatically formed (Z)-hinokiresinol obtained following incubation of p-coumaryl p-coumarate with a mixture of equal amounts of recZHRSa and recZHRSf) was found to be the optically pure (75)-isomer, which is identical to that isolated from A. officinalis cells (Table 12.1). A similar result was obtained with the crude plant protein from A. officinalis cultured cells, where the formed (Z)-hinokiresinol was almost optically pure, 97.2% e.e. in favor of the (75)-isomer (Table 12.1). In sharp contrast, when each subunit protein, recZHRSa or recZHRSP, was individually incubated with p-coumaryl p-coumarate, ( )-hinokiresinol was formed (Table 12.1). The enantiomeric compositions of ( )-hinokiresinol thus formed were 20.6% e.e. (with recZHRSa) and 9.0% e.e. (with recZHRSP) in favor of the (7S)-enantiomer (Table 12.1). Taken together, these results clearly indicate that the subunit composition of ZHRS controls not only cis/trans selectivity but also enantioselectivity in hinokiresinol formation (Fig. 12.3). This provides a novel example of enantiomeric control in the biosynthesis of natural products. Although the mechanism for the cis/trans selective and enantioselective reaction remains to be elucidated, for example by x-ray crystallography, the enantioselective mechanism totally differs from the enantioselectivity in biosynthesis of lignans, another class of phenylpropanoid compounds closely related to norlignans in terms of structure and biosynthesis. 

Umezawa T (2001) Biosynthesis of hgnans and related phenylpropanoid compounds. Regul Plant Growth Dev 36 57-67... 

Results of Wulf et al (7) show that carrot roots obtained from a supermarket contain myristicin Imperator variety carrots contain an average of 15 parts per million (ppm). Recently harvested, unprocessed carrots only rarely contain myristicin (8). The presence of myristicin in supermarket carrots and its absence in recently harvested ones indicate that its increased concentration may have been induced by some elicitor following harvest. Solar radiation after harvest, or fluorescent lighting during display, may function as such an elicitor. Light is known to produce ethylene and is an activator of phenylalanine ammonia-lyase, one of the regulatory enzymes responsible for phenylpropanoid biosynthesis in plants (9). 

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. 

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). 

Figure 1. Phenylalanine ammonia-lyase (PAL) involvement in the biosynthesis of phenylpropanoid-derived secondary metabolites in plants and Ba-sidiomycetes.

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. ... 

Enzymatic preparations for PAL from monocotyledonous species (monocots) can show a similar activity against tyrosine (tyrosine ammonia lyase, TAL), and TAL enzymatic preparations also show PAL activity. That a single enzyme may account for the observed cooccurring TAL and PAL activities was confirmed by Rosier et al., who showed the recombinant Zea mays (maize) PAL converted tyrosine to 4-coumarate directly, thus removing the requirement for the usual 4-hydroxylation step in phenylpropanoid biosynthesis. 

Flavanones are converted stereospecifically to the respective (2i ,3i )-dihydroflavonols (DHFs) by flavanone 3(3-hydroxylase. Stereospecificity for (2S)-flavanones has been confirmed by analysis of the recombinant protein. Flavanone 3(3-hydroxylase is commonly abbreviated to F3H, which is what has been used in this chapter, but FHT is also used in the literature, which agrees with the nomenclature for phenylpropanoid biosynthesis proposed in Heller and Forkmann. ... 

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