Ammonia lyase, phenylalanine

1 Phenylalanine Ammonia Lyase (PAL).- PAL usually exhibits negative subunit cooperativity, i.e. the binding of the first 

L-isomers are assumed to pack into the same active sites, though 

Many other compounds are involved in flavonoid biosynthesis in some species, for example, as donors for methylation or aromatic or aliphatic acylation. For intact plants, these are generally accepted to be available in the cell for the reaction to proceed if the appropriate modification activity is present. 

PAL is one of the best-characterized enzymes of plant secondary metabolism. It converts l-phenylalanine into tran -cinnamate ( -cinnamate) by the tra 5-elimination of ammonia and the pro-StS proton (see Ref. 4 for a full reaction discussion). The enzyme, which requires no cofactor, is a tetramer of 310-340 kDa. A cDNA for PAL was first isolated from Petrose-linum crispum (parsley), and others have subsequently been isolated from numerous species. Often PAL is produced from a multigene family and is present in a variety of isoenzyme forms. 

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. 

Considerable progress has been made with elucidating the functional aspects of the PAL protein, assisted by the availability of the crystal structure for histidine ammonia lyase (HAL), which catalyzes a reaction similar to that of PAL, the conversion of L-histidine to 

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Biosynthesis of Tea Flavonoids. The pathways for the de novo biosynthesis of flavonoids in both soft and woody plants (Pigs. 3 and 4) have been generally elucidated and reviewed in detail (32,51). The regulation and control of these pathways in tea and the nature of the enzymes involved in synthesis in tea have not been studied exhaustively. The key enzymes thought to be involved in the biosynthesis of tea flavonoids are 5-dehydroshikimate reductase (52), phenylalanine ammonia lyase (53), and those associated with the shikimate/arogenate pathway (52). At least 13 enzymes catalyze the formation of plant flavonoids (Table 4). 

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. 

L-Phenylalanine can be synthesised from trims-cinnamic add (Figure A8.12) catalysed by a L-phenylalanine ammonia-lyase from Rhodococcus glutinis. The commercialisation of the process was limited by the low conversion (70%), low stability of the biocatalyst and die severe inhibition exerted by trims-cinnamic add. These problems were largely overcome by researchers at Genex. The process, commercialised for a short period by Gen ex, involves a cell-free preparation of phenylalanine-ammonia-lyase activity from Rhodotorula rubra. 

Chappell, J., Hahlbrock, K. Boiler, T. (1984). Rapid induction of ethylene biosynthesis in cultured parsley cells by fungal elicitor and its relationship to the induction of phenylalanine ammonia-lyase. Planta, 161, 475-80. 

Kuhn, D.N., Chappell, J., Boudet, A. Hahlbrock, K. (1984). Induction of phenylalanine ammonia-lyase and 4-coumarate CoA ligase mRNAs in cultured plant cells by UV light or fungal elicitor. Proceedings of the National Academy of Sciences, USA, 81,1102-6. 

Lawton, M.A., Dixon, R.A., Rowell, P.M., Bailey, J.A. Lamb, C.J. (1983). Rapid induction of the synthesis of phenylalanine ammonia-lyase and of chalcone synthase in elicitor-treated plant cells. European Journal of Biochemistry, 129, 593-601. 

Fig. 12. Tentative model of the signal transduction chain that links the perception of pectic fragments to defense responses in carrot cells. Abbreviations apy, heterotrimeric G protein CaM, calmodulin 4CL, 4-coumarate-CoA ligase CTX, cholera toxin FC, fusicoccine GDP-P-S and GTP-y-S, guanosine 5 -0-(2-thiodiphosphate) and guanosine 5 -0-(3-thiotriphosphate) IP3, 1,4,5-inositol trisphosphate PAL, phenylalanine ammonia-lyase PLC, phospholipase C PR, pathogenesis related PTX, pertussis toxin Rc, receptor SP, staurosporine. Activation and inhibition are symbolized by + and -respectively.

Xiang L, BS Moore (2002) Inactivation, complementation, and heterologous expression of encP, a novel bacterial phenylalanine ammonia-lyase gene. J Biol Chem 277 32505-32509. 

Phenylalanine ammonia-lyase (PAL EC 4.3.1.5) is a pivotal enzyme in controlling flow of carbon from aromatic amino acids to secondary aromatic compounds (Figure 1) (28). PAL primarily deaminates phenylalanine to form t-cinnamic acid, however, in many species, it also less efficiently deaminates tyrosine to form -coumaric acid. Because PAL is restricted to plants and is an important enzyme in plant development, Jangaard (29) suggested that PAL inhibitors might make safe and effective herbicides, however, in his screen of several herbicides, he found no compound to have a specific effect on PAL. This was also the case in studies by Hoagland and Duke (30, 31.) in which 16 herbicides were screened. 

Figure 1. Schematic outline of various products and associated enzymes from the shikimate and phenolic pathways in plants (and some microorganisms). Enzymes (1) 3-deoxy-2-oxo-D-arabino-heptulosate-7-phosphate synthase (2) 5-dehydroquinate synthase (3) shikimate dehydrogenase (4) shikimate kinase (5) 5-enol-pyruvylshikimate-3-phosphate synthase (6) chorismate synthase (7) chorismate mutase (8) prephenate dehydrogenase (9) tyrosine aminotransferase (10) prephenate dehydratase (11) phenylalanine aminotransferase (12) anthranilate synthase (13) tryptophan synthase (14) phenylalanine ammonia-lyase (15) tyrosine ammonia-lyase and (16) polyphenol oxidase. (From ACS Symposium Series No. 181, 1982) (62).

Dehydroshikimate reductase and phenylalanine ammonia lyase mediate reactions involved in the synthesis of the polyphenols and are therefore key components of tea leaf.24... 

Inhibition of synthesis of the aromatic matrix by inhibitors of phenylalanine ammonia lyase causes the inhibition of deposition of aliphatic components and prevents development of diffusion resistance. Inhibition of synthesis of peroxidase, the enzyme involved in the deposition of the polymeric phenolic matrix, caused by iron deficiency, prevents deposition of aliphatic components of suberin. 

Wieder, K.J., Palczuk, N.C., van Es, T., and Davis, F.F. (1979) Some properties of polyethylene glycol Phenylalanine ammonia-lyase adducts./. Biol. Chem. 254, 12579-12587. 

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. 

In the first enzymatic step, phenylalanine ammonia lyase (PAL) converts phenylalanine to trans cinnamate, via a deamination reaction liberating ammonia. PAL can also convert tyrosine to p-coumarate, albeit at lower efficiency (MacDonald and D Cunha 2007). PAL functions as a tetramer of identical subunits, with two subunits combining to form one active site (Stafford 1990 MacDonald and D Cunha 2007). 

Figure 5.4. Abbreviated scheme for biosynthesis of major flavonoid subclasses, showing the primary enzymes and substrates leading to different subclasses. Bold-faced, uppercase abbreviations refer to enzyme names, whereas substrate names are presented in lowercase letters. PAL, phenylalanine ammonia lyase C4H, cinnamate 4-hydroxylase 4CL, 4-coumarate CoA ligase CHS, chalcone synthase CHI, chalcone isomerase CHR, chalcone reductase IPS, isoflavone synthase F3H, flavonone 3-hydroxylase F3 H, flavonoid 3 -hydroxylase F3 5 H, flavonoid 3 5 -hydroxylase FNSI/II, flavone synthase DFR, dihydroflavonol 4-reductase FLS, flavonol synthase ANS, anthocyanidin synthase LAR, leucoanthocyanidin reductase ANR, anthocyanidin reductase UFGT, UDP-glucose flavonoid 3-O-glucosyltransferase. R3 = H or OH. R5 = H or OH. Glc = glucose. Please refer to text for more information.

MacDonald MJ and D Cunha GB. 2007. A modern view of phenylalanine ammonia lyase. Biochem Cell Biol 85 273-282. 

Figure 6.1 Major branch pathways of flavonoid biosynthesis in Arabidopsis. Branch pathways, enzymes, and end products present in other plants but not Arabidopsis are shown in light gray. Abbreviations cinnamate-4-hydroxylase (C4H), chalcone isomerase (CHI), chalcone synthase (CHS), 4-coumarate CoA-ligase (4CL), dihydroflavonol 4-reductase (DFR), flavanone 3-hydroxylase (F3H), flavonoid 3 or 3 5 hydroxylase (F3 H, F3 5 H), leucoanthocyanidin dioxygenase (LDOX), leucoanthocyanidin reductase (LCR), O-methyltransferase (OMT), phenylalanine ammonia-lyase (PAL), rhamnosyl transferase (RT), and UDP flavonoid glucosyl transferase (UFGT).

Calabrese JC, Jordan DB, Boodhoo A, Sariaslani S, Vannelli T (2004) Crystal structure of phenylalanine ammonia lyase multiple helix dipoles implicated in catalysis. Biochemistry 43(36) 11403-11416... 

Ritter H, Schulz GE (2004) Structural basis for the entrance into the phenylpropanoid metabolism catalyzed by phenylalanine ammonia-lyase. Plant Cell 16(12) 3426-3436... 

Maier VP, Hasegawa S (1970) L-Phenylalanine ammonia-lyase activity and naringenin glycoside accumulation in developing grapefruit. Phytochemistry 9 139-144... 

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