Phenylalanine-ammonium lyase

ENZ enzyme assays, SC structural composition, MM molecular methods, IL isotopic labeling, IF isotopic fractionation, INH inhibition studies, UNK unknown, LOX lipoxogenase, EPSP synthase 5-enolpyruvylshikimate-3-phosphate, SDH shikimate dehydrogenase, PAL phenylalanine ammonium lyase, PKS polyketide synthase, NRPS nonribosomal peptide synthase 1 Gerwick 1999 2 Liu et al. 1994 3 Boonprab et al. 2003 4 Cvejic and Rohmer 1999 5 Disch et al. 1998 6 Chikaraishi et al. 2006 7 Schwender et al. 2001 8 Schwender et al. 1997 9 Mayes et al. 1993 10 Shick et al. 1999 11 Richards et al. 2006 12 Bouarab et al. 2004 13 Pelletreau et al., unpublished data 14 Dittman and Weigand 2006 15 Rein and Barrone 1999 Empty columns imply no direct evidence of these enzymes from these systems... 

The biosynthetic pathway for salicylic acid is not clear. At present, at least two pathways have been proposed. Each branches from phenyl-propanoid biosynthesis after phenylalanine has been converted to trans-cinnamic acid by phenylalanine ammonium lyase (PAL). In one scheme (Pathway 1 Fig. 4), tram-cinnamic acid would be converted to 2-hydroxy cinnamic acid (or 2-coumaric acid) by a cinnamate 2-hydroxylase. This compound could then be converted to salicylic acid via -oxidation possibly through an acetyl coenzyme A (CoA) intermediate. Alternatively, tram-cinnamic acid could be oxidized to benzoic acid and then hydrox-ylated via a postulated o-hydroxylase activity. The details of this pathway, particularly in tobacco and cucumber, deserve further study. 

In either of the proposed pathways, salicylic acid is synthesised from tram-cinnamic acid. This is an intriguing observation and may provide a clue as to how and why the induction of SAR is tightly linked to the formation of a necrotic lesion. When plants react hypersensitively to pathogen attack, many biochemical changes occur, including the induction of phenylpropanoid biosynthesis. In bean, as well as other plants, this induction seems to be at least partly caused by an increase in the synthesis of phenylalanine ammonium lyase and other enzymes involved in the biosynthesis of isoflavonoid phytoalexins, flavonoid pigments and... 

First let us mention the biosynthesis of the cinnamic acids themselves (Fig. 96). As mentioned earlier they are derived from phenylalanine and tyrosine. By oxidative deamination phenylalanine is converted to cinnamic acid and tyrosine to / -coumaric acid. Since ammonia in the form of ammonium ions is set free in this reaction the enzymes concerned are called ammonium lyases. The tyrosine-ammonium lyase seems to be particularly important in grasses but is also to be found in the rest of the plant kingdom. However, the phenylalanine-ammonium lyase (PAL) is the more important of these two enzymes. We shall come across it again as the key enzyme of phenylpropane synthesis. 

Fig. 96. The biosynthesis of cinnamic acids. 1 = Phenylalanine-ammonium-lyase, 2 = Tyrosine-ammonium-lyase.

Anthocyanin synthesis is a very complicated process in which many genes are involved (page 132). Various authors Such as Zucker, Mohr, and Zenk discovered in the 1960s that light is capable of stimulating the synthesis of the phenylalanine-ammonium-lyase via the phytochrome system. Thus, light activates the genetic material for the synthesis of a key... 

Let us call to mind the key enzyme of phenylpropane metabolism, phenylalanine-ammonium-lyase or PAL (page 122). Its synthesis can be induced by light in a variety of plants, including cucumber seedlings. One of the products resulting from PAL activity, / -coumaric acid, can repress the synthesis of the enzyme. The nature of this repression is complex. In one sense it appears to be end product repression in accordance with the Jacob-Monod model, but there is also circumstantial evidence for the participation of a newly formed protein. However it may be, the accumula-... 

Cinnamic acid (48) is synthesized universally in higher plants and widely in fungi from (25)-phenylalanine by phenylalanine ammonium lyase. This process is a trans elimination of the elements of ammonia, with stereospecific loss of the 3-pro S) proton of phenylalanine (46). Phenylpyruvic acid (49) (shown in its enol form) is the normal biogenetic precursor of phenylalanine, and is in equilibrium with it by means of the action of aminotransferases and amino acid oxidases (see Fig. 11). To distinguish between the participation of cinnamic acid (48) and phenylpyruvic acid (49) and to clarify the mechanism involved in the proton losses, (2R,35)-[3- H]-, (2S,3R)-[3- H], and (25)-[f/- C]phenylalanines were administered to the cultures. The incorporations resulted in the removal of 57% and 76%, respectively, of the labeled hydrogen. 

One of the most interesting uses for cinnamic acid in recent years has been as a raw material in the preparation of L-phenylalanine [63-91-2] the key intermediate for the synthetic dipeptide sweetener aspartame (25). Genex has described a biosynthetic route to L-phenylalanine which involves treatment of immobilized ceUs of R rubra containing the enzyme phenylalanine ammonia lyase (PAT,) with ammonium cinnamate [25459-05-6] (26). 

The first step of phenylpropanoid biosynthesis is conversion of phenylalanine into cinnamic acid by cleavage of ammonium group by the enzyme phenylalanine ammonia-lyase (PAL). Reduction of carboxylic acid from the cinnamic acid leads to cinnamaldehyde, which is then acylated with acetate from acetyl-CoA to form coniferyl alcohol [14]. Reductive cleavage of coniferyl alcohol by eugenol synthase yields eugenol [15]. 

Applications of ammonia lyases Ammonia lyases catalyze the reversible, regio- and stereoselective addition of ammonia to alkenes. The family of lyases includes aspartate, methyl aspartate, histidine, tyrosine and phenylalanine ammonia lyases. The system consisting of phenylalanine ammonia lyase (PAL, EC 4.3.5.1), cinnamic acid, water and ammonium ions was optimized in terms of pH, temperature, reaction time, concentration and buffer for the synthesis of (2S,3S)- and (25,37 )-[3- H]phenylalanines. 

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