Enzymes L-phenylalanine-ammonia lyase

Catabolism of histidine in most organisms proceeds via an initial elimination of NH3 to form urocanic acid (Eq. 14-44). The absence of the enzyme L-histidine ammonia-lyase (histidase) causes the genetic disease histidinemia 284/285 A similar reaction is catalyzed by the important plant enzyme L-phenylalanine ammonia-lyase. It eliminates -NH3+ along with the pro-S hydrogen in the (3 position of phenylalanine to form frans-cinnamate (Eq. 14-45). Tyrosine is converted to p-coumarate by the same enzyme. Cinnamate and coumarate are formed in higher plants and are converted into a vast array of derivatives (Box 21-E,... 

Phenylalanine is converted to cinnamic acid by the action of the ubiquitous enzyme L-phenylalanine ammonia lyase (PAL) (EC 4.3.11.5) (Davin et al., 1992 Hanson and Havir, 1981). This enzyme, which catalyzes loss of ammonia and production of -cinnamic acid (1), has been detected in a large number of vascular plants and several genera of basidi-omycetes. PAL occurs mainly in the cytoplasm, but some plants contain low levels of PAL in plastids, mitochondria, and microbodies (Hanson and Havir, 1981). Cinnamate is produced by the action of PAL in an elimination reaction... 

In higher plants the polymer, lignin, and various aromatic secondary metabolites, notably many alkaloids (Chapter 6) and flavonoids (Section 5.4) are formed from the aromatic amino acids, L-phenylalanine (5.77) and/or L-tyrosine (5.75). [For some alkaloids as well as some microbial metabolites, tryptophan (5.7 ) is the source of their particular aromatic rings.] There are for these metabolites common pathways leading from phenylalanine, and in some plants tyrosine, to phenylpropanoid (Cg-Cg) intermediates. The first step from phenylalanine involves the enzyme L-phenylalanine ammonia lyase (known, perhaps affectionately, as PAL), an enzyme widely distributed, and well-characterized. Elimination of ammonia occurs to give cinnamic acid 5.23). It involves loss of the (3-/ ro-5)-proton of L-phenylalanine (5.77), and thus occurs in the aw z-sense [L-tyrosine ammonia lyase functions to remove also the (3-/ ro-5)-proton in tyrosine] [12, 13]. 

The enzyme L-phenylalanine ammonia lyase has been isolated and extensively purified from potato tuber , com , the yeast Rhodotorula and the procaryotic organism Strepto-... 

Laluente MT, Sala JM, Zacarias L (2004) Active oxygen detoxifying enzymes and phenylalanine ammonia-lyase in the ethylene-induced chilling tolerance in citrus fmit. J Agric Food Chem 52(11) 3606-3611... 

L-Phenylalanine ammonia lyase (PAL EC 4.3.1.5), an enzyme found in a variety of plants, catalyzes both the deamination of L-phenylalanine to (L )-3-phenyl-2-butenoic acid and the reverse reaction33-36. 

Biosynthesis. The primary precursors of L. are con-iferyl, sinapyl and p-coumaiyl alcohoi, which are derived from 4-hydroxycinnamic acid. L. from conifers (i. e. from softwood) is derived chiefly from conifeiyl alcohol with variable but small proportions of sinapyl and p-coumaryl alcohol. L. from dicotyledonous an-giosperms (i.e. from hardwood), particularly deciduous trees, is formed chiefly from sinapyl (-44%) and coniferyl (-48 %) alcohol, with about 8 % p-cou-maryl alcohol. L. in grasses is formed from p-coumar-yl (-30%), coniferyl (-50%) and sinapyl (-20%) alcohol. These primary L. precursors are formed from the aromatic amino acids L-phenylalanine and L-tyro-sine by a series of reactions shown (Rg.). The first reaction is catalysed by L-Phenylalanine ammonia-lyase (EC 4.3.1.5) (see) this enzyme is induced by light in a process involving phytochrome, and it is of general importance in the synthesis of plant phenolic compounds from phenylalanine and tyrosine. 

The aromatic amino acid L-phenylalanine (primary metabolite) is directed into the phe-nylpropanoid pathway leading to hydroxy-cinnamic acids, lignin and flavonoids by the activity of L-phenylalanine ammonia-lyase (PAL), which brings about its nonoxidative deamination yielding ammonia and tvans-cinnamic acid (Fig. 1). PAL is one of the most studied plant enzymes, and its crystal structure has recently been solved [2]. PAL is related to the histidine and tyrosine ammonia-lyases of amino acid catabolism. A class of bifunctional PALs found in monocotyle-donous plants and yeast can also deaminate tyrosine [3]. A single His residue is responsible for this switch in substrate preference [3, 4]. All three enzymes share a unique MIO (4-methylidene-imidazole-5-one) prosthetic group at the active site. This is formed auto-catalytically from the tripeptide Ala-Ser-Gly by cyclization and dehydration during a late... 

It is reasonable to assume from the available evidence that the enzyme acts at a switching point in metabolism and diverts L-phenylalanine from the general pool of amino acids used for protein synthesis to the biosynthesis of phenylpropanoid compounds. Since initial steps are probable sites for overall pathway regulation, it is therefore not surprising that the factors which influence L-phenylalanine ammonia lyase activity have been subject to detailed scrutiny. Thus phytochrome control in dark grown seedlings. 

Chemical inhibition of L-phenylalanine ammonia lyase activity may be achieved by the use of typical carbonyl reagents such as sodium borohydride and potassium cyanide. Treatment of the enzyme with tritiated sodium borohydride and subsequent hydrolysis gave alanine in which the majority of the radioactivity was confined to the jj-methyl group . Similarly reaction with potassium cyanide and hydrolysis gave aspartic acid labelled exclusively in the -carboxyl group . These observations led to the proposal that the active site of the enzyme, like that of the related L-histidine ammonia lyase , contains a dehydro-alanine residue... 

Dehydroalanine units have been identified at the active sites of the enzymes histidine ammonia lyase 144,434) and L-phenylalanine ammonia lyase (164), either by reduction with NaB Ht (164, 434), or by addition of nitromethane (144) and subsequent reduction to the amine. Following hydrolysis it was possible to detect tritiated alanine or a,y-diamino-butyric acid respectively. 

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). 

Reaction 1 is governed by the enzyme phenylalanine ammonia lyase. This enzyme normally conducts the breakdown of L-phenylalanine to from-cinnamic add and ammonia. However, die reaction can be reversed leading to the production of L-phenylalanine from frans-dnnamic add by using excess ammonia. 

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 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. 

Lyases are an attractive group of enzymes from a commercial perspective, as demonstrated by then-use in many industrial processes.240 They catalyze the cleavage of C-C, C-N, C-O, and other bonds by means other than hydrolysis, often forming double bonds. For example, two well-studied ammonia lyases, aspartate ammonia lyase (aspartase) (E.C. 4.3.1.1) and phenylalanine ammonia lyase (PAL) (E.C. 4.3.1.5), catalyze the trans-elimination of ammonia from the amino acids, l-aspartate and L-phenylalanine, respectively. Most commonly used in the synthetic mode, the reverse reaction has been used to prepare the L-amino acids at the ton scale (Schemes 19.30 and 19.31).240 242 These reactions are conducted at very high substrate concentrations such that the equilibrium is shifted, resulting in very high conversion to the amino acid products. 

Various commercial routes for the production of L-phenyalanine have been developed because of the utilization of this amino acid in the dipeptide sweetener Aspartame. One route that has been actively pursued is the synthesis of L-phenylalanine from trans-cinnamic acid using the enzyme phenylalanine ammonia lyase (105,106). This enzyme catalyzes the reversible, nonoxidative deamination of L-phenylalanine and can be isolated from various plant and microbial sources (107,108). 

As can be seen from Table 8.7 productivity (expressed in g h b is highest for precursor addition. The production of L-phenylalanine from phenylpyruvic add also has the shortest reaction time to obtain hi conversions. The pH commonly used is around 75, quite normal for biological processes. Only the enzyme phenylalanine ammonia lyase shows an optimiim pH of lO.The process temperature varies between 30 and 40°C with an average of 35°C. No extreme temperatures have been reported due to the fact that denaturation occurs at hi temperatures. The optimal concentration for cells frequently used is 10-20 g 1". However, conversion of ACA is done with hi cell mass concentrations in recent studies possibly to compensate for substrate inhibition and thus to maintain hi product concentration. The processes using PPA and ACA need an amino add as amino donor, usually L-aspartic add is used. 

L-Phenylalanine,which is derived via the shikimic acid pathway,is an important precursor for aromatic aroma components. This amino acid can be transformed into phe-nylpyruvate by transamination and by subsequent decarboxylation to 2-phenylacetyl-CoA in an analogous reaction as discussed for leucine and valine. 2-Phenylacetyl-CoA is converted into esters of a variety of alcohols or reduced to 2-phenylethanol and transformed into 2-phenyl-ethyl esters. The end products of phenylalanine catabolism are fumaric acid and acetoacetate which are further metabolized by the TCA-cycle. Phenylalanine ammonia lyase converts the amino acid into cinnamic acid, the key intermediate of phenylpropanoid metabolism. By a series of enzymes (cinnamate-4-hydroxylase, p-coumarate 3-hydroxylase, catechol O-methyltransferase and ferulate 5-hydroxylase) cinnamic acid is transformed into p-couma-ric-, caffeic-, ferulic-, 5-hydroxyferulic- and sinapic acids,which act as precursors for flavor components and are important intermediates in the biosynthesis of fla-vonoides, lignins, etc. Reduction of cinnamic acids to aldehydes and alcohols by cinnamoyl-CoA NADPH-oxido-reductase and cinnamoyl-alcohol-dehydrogenase form important flavor compounds such as cinnamic aldehyde, cin-namyl alcohol and esters. Further reduction of cinnamyl alcohols lead to propenyl- and allylphenols such as... 

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