Phenylalanine ammonia-lyase

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phenylalanine ammonia-lyase
Phenylalanine ammonia lyase.png
PDB rendering based on 1T6J.
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EC no. 4.3.1.24
CAS no. 9024-28-6
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The enzyme phenylalanine ammonia lyase (EC 4.3.1.24) catalyzes the conversion of L-phenylalanine to ammonia and trans-cinnamic acid.: [1]

Contents

L-phenylalanine = trans-cinnamate + NH3

Phenylalanine ammonia lyase (PAL) is the first and committed step in the phenyl propanoid pathway and is therefore involved in the biosynthesis of the polyphenol compounds such as flavonoids, phenylpropanoids, and lignin in plants. [2] [3] Phenylalanine ammonia lyase is found widely in plants, as well as some bacteria, yeast, and fungi, with isoenzymes existing within many different species. It has a molecular mass in the range of 270–330  kDa. [1] [4] The activity of PAL is induced dramatically in response to various stimuli such as tissue wounding, pathogenic attack, light, low temperatures, and hormones. [1] [5] PAL has recently been studied for possible therapeutic benefits in humans afflicted with phenylketonuria. [6] It has also been used in the generation of L-phenylalanine as precursor of the sweetener aspartame. [7]

The enzyme is a member of the ammonia lyase family, which cleaves carbon–nitrogen bonds. Like other lyases, PAL requires only one substrate for the forward reaction, but two for the reverse. It is thought to be mechanistically similar to the related enzyme histidine ammonia-lyase (EC:4.3.1.3, HAL). [8] The systematic name of this enzyme class is L-phenylalanine ammonia-lyase (trans-cinnamate-forming). Previously, it was designated as EC 4.3.1.5, but that class has been redesignated as EC 4.3.1.24 (phenylalanine ammonia-lyases), EC 4.3.1.25 (tyrosine ammonia-lyases), and EC 4.3.1.26 (phenylalanine/tyrosine ammonia-lyases). Other names in common use include tyrase, phenylalanine deaminase, tyrosine ammonia-lyase, L-tyrosine ammonia-lyase, phenylalanine ammonium-lyase, PAL, and L-phenylalanine ammonia-lyase.

Mechanism

Phenylalanine ammonia lyase is specific for L-phenylalanine, and to a lesser extent, L-tyrosine. [9] [10] The reaction catalyzed by PAL is a spontaneous elimination reaction rather than an oxidative deamination. [11]

The cofactor 3,5-dihydro-5-methyldiene-4H-imidazol-4-one (MIO) is involved in the reaction and sits atop the positive pole of three polar helices in the active site, which helps to increase its electrophilicity. [12] MIO is attacked by the aromatic ring of L-phe, which activates the C-H bond on the β carbon for deprotonation by a basic residue. [13] [14] The carbanion intermediate of this E1cB-elimination reaction, which is stabilized by partial positive regions in the active site, then expels ammonia to form the cinnamate alkene. The mechanism of the reaction of PAL is thought to be similar to the mechanism of the related enzyme histidine ammonia lyase. [13]

Proposed autocatalytic formation of MIO cofactor from the tripeptide Ala-Ser-Gly by two water elimination steps. MIO.png
Proposed autocatalytic formation of MIO cofactor from the tripeptide Ala-Ser-Gly by two water elimination steps.

A dehydroalanine residue was long thought to be the key electrophilic catalytic residue in PAL and HAL, but the active residue was later found instead to be MIO, which is even more electrophilic. [16] [17] It is formed by cyclization and dehydration of conserved Ala-Ser-Gly tripeptide segment. The first step of MIO formation is a cyclization-elimination by an intramolecular nucleophilic attack of the nitrogen of Gly204 at the carbonyl group of Ala202. A subsequent water elimination from the side chain of Ser203 completes the system of crossconjugated double bonds. [15] Numbers are given for the phenylalanine ammonia lyase from Petroselinumcrispum (PDB 1W27). Although MIO is a polypeptide modification, it was proposed to call it a prosthetic group, because it has the quality of an added organic compound. [8]

PAL is inhibited by trans-cinnamic acid, and, in some species, may be inhibited by trans-cinnamic acid derivatives. [1] [18] The unnatural amino acids D-Phe and D-Tyr, the enantiomeric forms of the normal substrate, are competitive inhibitors. [9]

Structure

PAL active site Phenylalanine ammonia lysase active site.png
PAL active site

Phenylalanine ammonia lyase is composed of four identical subunits composed mainly of alpha-helices, with pairs of monomers forming a single active site. [17] Catalysis in PAL may be governed by the dipole moments of seven different alpha helices associated with the active site. [19] The active site contains the electrophilic group MIO non-covalently bonded to three helices. Leu266, Asn270, Val269, Leu215, Lys486, and Ile472 are located on the active site helices, while Phe413, Glu496, and Gln500 contribute to the stabilization of the MIO cofactor. The orientation of dipole moments generated by helices within the active site generates an electropositive region for ideal reactivity with MIO. The partially positive regions in the active site may also help stabilize the charge of a carbanion intermediate. PAL is structurally similar to the mechanistically related histidine ammonia lyase, although PAL has approximately 215 additional residues. [17]

Function

Phenylalanine ammonia lyase can perform different functions in different species. It is found mainly in some plants and fungi (i.e. yeast). In fungal and yeast cells, PAL plays an important catabolic role, generating carbon and nitrogen. [2] In plants it is a key biosynthetic enzyme that catalyzes the first step in the synthesis of a variety of polyphenyl compounds [2] [3] and is mainly involved in defense mechanisms. PAL is involved in 5 metabolic pathways: tyrosine metabolism, phenylalanine metabolism, nitrogen metabolism, phenylpropanoid biosynthesis, and alkaloid biosynthesis.

Disease relevance

Enzyme substitution therapy using PAL to treat phenylketonuria (PKU), an autosomal recessive genetic disorder in humans in which mutations in the phenylalanine hydroxylase (PAH, EC 1.14.16.1) gene inactivate the enzyme is being explored. [6] This leads to an inability of the patient to metabolize phenylalanine, causing elevated levels of Phe in the bloodstream (hyperphenylalaninemia) and mental retardation if therapy is not begun at birth. [6]

In May 2018, the FDA approved pegvaliase, a recombinant PEGylated phenylalanine ammonia-lyase for the treatment of PKU that had been developed by Biomarin. [20] [21]

In plants

Lactuca sativa was investigated by Vàsquez et al. 2017. They find that UV-C treatment increased PAL enzyme activity. This increase results in decreased susceptibility to Botrytis cinerea . [22]

Research

Artificial sweeteners

The reverse reaction catalyzed by PAL has been explored for use to convert trans-cinnamic acid to L-phenylalanine, which is a precursor of the sweetener aspartame. This process was developed by Genex Corporation but was never commercially adopted. [23]

Unnatural amino acid synthesis

Analogous to how aspartame is synthesized, PAL is also used to synthesize unnatural amino acids from various substituted cinnamic acids for research purposes. [24] Steric hindrance from arene substitution limits PAL's utility for this purpose however. [25] For instance, when Rhodotorula glutinis was used to affect this biotransformation the enzyme was discovered to be intolerant of all para substituents other than fluorine, presumably due to the element's small atomic radius. Meta and ortho positions were found to be more tolerant, but still limited by, larger substituents. For instance the enzyme's active site permitted ortho methoxy substitution but forbade meta ethoxy. Other organisms with different versions of the enzyme may be less limited in this way. [26] [27]

Structural studies

As of late 2007, 5 structures have been solved for this class of enzymes, with PDB accession codes 1T6J, 1T6P, 1W27, 1Y2M, and 2NYF.

Related Research Articles

<span class="mw-page-title-main">Tyrosine</span> Amino acid

L-Tyrosine or tyrosine or 4-hydroxyphenylalanine is one of the 20 standard amino acids that are used by cells to synthesize proteins. It is a non-essential amino acid with a polar side group. The word "tyrosine" is from the Greek tyrós, meaning cheese, as it was first discovered in 1846 by German chemist Justus von Liebig in the protein casein from cheese. It is called tyrosyl when referred to as a functional group or side chain. While tyrosine is generally classified as a hydrophobic amino acid, it is more hydrophilic than phenylalanine. It is encoded by the codons UAC and UAU in messenger RNA.

<span class="mw-page-title-main">Phenylalanine</span> Type of α-amino acid

Phenylalanine is an essential α-amino acid with the formula C
9H
11NO
2. It can be viewed as a benzyl group substituted for the methyl group of alanine, or a phenyl group in place of a terminal hydrogen of alanine. This essential amino acid is classified as neutral, and nonpolar because of the inert and hydrophobic nature of the benzyl side chain. The L-isomer is used to biochemically form proteins coded for by DNA. Phenylalanine is a precursor for tyrosine, the monoamine neurotransmitters dopamine, norepinephrine (noradrenaline), and epinephrine (adrenaline), and the biological pigment melanin. It is encoded by the messenger RNA codons UUU and UUC.

<span class="mw-page-title-main">Phenylalanine hydroxylase</span> Mammalian protein found in Homo sapiens

Phenylalanine hydroxylase (PAH) (EC 1.14.16.1) is an enzyme that catalyzes the hydroxylation of the aromatic side-chain of phenylalanine to generate tyrosine. PAH is one of three members of the biopterin-dependent aromatic amino acid hydroxylases, a class of monooxygenase that uses tetrahydrobiopterin (BH4, a pteridine cofactor) and a non-heme iron for catalysis. During the reaction, molecular oxygen is heterolytically cleaved with sequential incorporation of one oxygen atom into BH4 and phenylalanine substrate. In humans, mutations in its encoding gene, PAH, can lead to the metabolic disorder phenylketonuria.

<span class="mw-page-title-main">Dehydroalanine</span> Chemical compound

Dehydroalanine is a dehydroamino acid. It does not exist in its free form, but it occurs naturally as a residue found in peptides of microbial origin. As an amino acid residue, it is unusual because it has an unsaturated backbone.

<span class="mw-page-title-main">Cinnamaldehyde</span> Chemical compound

Cinnamaldehyde is an organic compound with the formula or C₆H₅CH=CHCHO. Occurring naturally as predominantly the trans (E) isomer, it gives cinnamon its flavor and odor. It is a phenylpropanoid that is naturally synthesized by the shikimate pathway. This pale yellow, viscous liquid occurs in the bark of cinnamon trees and other species of the genus Cinnamomum. The essential oil of cinnamon bark is about 90% cinnamaldehyde. Cinnamaldehyde decomposes to styrene because of oxidation as a result of bad storage or transport conditions. Styrene especially forms in high humidity and high temperatures. This is the reason why cinnamon contains small amounts of styrene.

Shikimic acid, more commonly known as its anionic form shikimate, is a cyclohexene, a cyclitol and a cyclohexanecarboxylic acid. It is an important biochemical metabolite in plants and microorganisms. Its name comes from the Japanese flower shikimi, from which it was first isolated in 1885 by Johan Fredrik Eykman. The elucidation of its structure was made nearly 50 years later.

<span class="mw-page-title-main">Apigenin</span> Chemical compound

Apigenin (4′,5,7-trihydroxyflavone), found in many plants, is a natural product belonging to the flavone class that is the aglycone of several naturally occurring glycosides. It is a yellow crystalline solid that has been used to dye wool.

<span class="mw-page-title-main">Phenylpropanoid</span>

The phenylpropanoids are a diverse family of organic compounds that are biosynthesized by plants from the amino acids phenylalanine and tyrosine in the shikimic acid pathway. Their name is derived from the six-carbon, aromatic phenyl group and the three-carbon propene tail of coumaric acid, which is the central intermediate in phenylpropanoid biosynthesis. From 4-coumaroyl-CoA emanates the biosynthesis of myriad natural products including lignols, flavonoids, isoflavonoids, coumarins, aurones, stilbenes, catechin, and phenylpropanoids. The coumaroyl component is produced from cinnamic acid.

<span class="mw-page-title-main">Aromatic amino acid</span> Amino acid having an aromatic ring

An aromatic amino acid is an amino acid that includes an aromatic ring.

<span class="mw-page-title-main">Histidine ammonia-lyase</span>

Histidine ammonia-lyase is an enzyme that in humans is encoded by the HAL gene. It converts histidine into ammonia and urocanic acid. Its systematic name is L-histidine ammonia-lyase (urocanate-forming).

<span class="mw-page-title-main">Cystathionine beta-lyase</span> Enzyme

Cystathionine beta-lyase, also commonly referred to as CBL or β-cystathionase, is an enzyme that primarily catalyzes the following α,β-elimination reaction

<span class="mw-page-title-main">Threonine ammonia-lyase</span>

Threonine ammonia-lyase (EC 4.3.1.19, systematic name L-threonine ammonia-lyase (2-oxobutanoate-forming), also commonly referred to as threonine deaminase or threonine dehydratase, is an enzyme responsible for catalyzing the conversion of L-threonine into α-ketobutyrate and ammonia:

<span class="mw-page-title-main">Arogenate dehydratase</span> Enzyme

Arogenate dehydratase (ADT) (EC 4.2.1.91) is an enzyme that catalyzes the chemical reaction

<span class="mw-page-title-main">Chorismate synthase</span>

The enzyme chorismate synthase catalyzes the chemical reaction

In enzymology, glutamate-prephenate aminotransferase is an enzyme that catalyzes the chemical reaction

<span class="mw-page-title-main">Rosavin</span> Chemical compound

Rosavin are a family of cinnamyl mono- and diglycosides that are key ingredients of Rhodiola rosea L.,. R. rosea is an important medicinal plant commonly used throughout Europe, Asia, and North America, that has been recognized as a botanical adaptogen by the European Medicines Agency. Rosavin production is specific to R. rosea and R. sachalinenis, and the biosynthesis of these glycosides occurs spontaneously in Rhodiola roots and rhizomes. The production of rosavins increases in plants as they get older, and the amount of the cinnamyl alcohol glycosides depends on the place of origin of the plant.

<span class="mw-page-title-main">Biopterin-dependent aromatic amino acid hydroxylase</span>

Biopterin-dependent aromatic amino acid hydroxylases (AAAH) are a family of aromatic amino acid hydroxylase enzymes which includes phenylalanine 4-hydroxylase, tyrosine 3-hydroxylase, and tryptophan 5-hydroxylase. These enzymes primarily hydroxylate the amino acids L-phenylalanine, L-tyrosine, and L-tryptophan, respectively.

The biosynthesis of phenylpropanoids involves a number of enzymes.

<span class="mw-page-title-main">Tyrosine ammonia-lyase</span>

Tyrosine ammonia lyase (EC 4.3.1.23, L-tyrosine ammonia-lyase, TAL or Tyrase) is an enzyme in the natural phenols biosynthesis pathway. It transforms L-tyrosine into p-coumaric acid.

Phenylalanine/tyrosine ammonia-lyase (EC 4.3.1.25, PTAL, bifunctional PAL) is an enzyme with systematic name L-phenylalanine(or L-tyrosine):trans-cinnamate(or trans-p-hydroxycinnamate) ammonia-lyase. This enzyme catalyses the following chemical reaction

References

  1. 1 2 3 4 Camm EL, Towers G (1 May 1973). "Phenylalanine ammonia lyase". Phytochemistry. 12 (5): 961–973. doi:10.1016/0031-9422(73)85001-0.
  2. 1 2 3 Fritz RR, Hodgins DS, Abell CW (August 1976). "Phenylalanine ammonia-lyase. Induction and purification from yeast and clearance in mammals". The Journal of Biological Chemistry. 251 (15): 4646–50. doi: 10.1016/S0021-9258(17)33251-9 . PMID   985816.
  3. 1 2 Tanaka Y, Matsuoka M, Yamanoto N, Ohashi Y, Kano-Murakami Y, Ozeki Y (August 1989). "Structure and characterization of a cDNA clone for phenylalanine ammonia-lyase from cut-injured roots of sweet potato". Plant Physiology. 90 (4): 1403–7. doi:10.1104/pp.90.4.1403. PMC   1061903 . PMID   16666943.
  4. ↑ Appert C, Logemann E, Hahlbrock K, Schmid J, Amrhein N (October 1994). "Structural and catalytic properties of the four phenylalanine ammonia-lyase isoenzymes from parsley (Petroselinum crispum Nym.)". European Journal of Biochemistry. 225 (1): 491–9. doi: 10.1111/j.1432-1033.1994.00491.x . PMID   7925471.
  5. ↑ Hahlbrock K, Grisebach H (1 June 1979). "Enzymic Controls in the Biosynthesis of Lignin and Flavonoids". Annual Review of Plant Physiology. 30 (1): 105–130. doi:10.1146/annurev.pp.30.060179.000541.
  6. 1 2 3 Sarkissian CN, Gámez A (December 2005). "Phenylalanine ammonia lyase, enzyme substitution therapy for phenylketonuria, where are we now?". Molecular Genetics and Metabolism. 86 (Suppl 1): S22-6. doi:10.1016/j.ymgme.2005.06.016. PMID   16165390.
  7. ↑ Evans C, Hanna K, Conrad D, Peterson W, Misawa M (1 February 1987). "Production of phenylalanine ammonia-lyase (PAL): isolation and evaluation of yeast strains suitable for commercial production of L-phenylalanine". Applied Microbiology and Biotechnology. 25 (5): 406–414. doi:10.1007/BF00253309. S2CID   40066810.
  8. 1 2 Schwede TF, Rétey J, Schulz GE (April 1999). "Crystal structure of histidine ammonia-lyase revealing a novel polypeptide modification as the catalytic electrophile". Biochemistry. 38 (17): 5355–61. doi:10.1021/bi982929q. PMID   10220322.
  9. 1 2 Hodgins DS (May 1971). "Yeast phenylalanine ammonia-lyase. Purification, properties, and the identification of catalytically essential dehydroalanine". The Journal of Biological Chemistry. 246 (9): 2977–85. doi: 10.1016/S0021-9258(18)62279-3 . PMID   5102931.
  10. ↑ Barros J, Serrani-Yarce JC, Chen F, Baxter D, Venables BJ, Dixon RA (May 2016). "Role of bifunctional ammonia-lyase in grass cell wall biosynthesis". Nature Plants. 2 (6): 16050. doi:10.1038/nplants.2016.50. PMID   27255834. S2CID   3462127.
  11. ↑ Koukol J, Conn EE (October 1961). "The metabolism of aromatic compounds in higher plants. IV. Purification and properties of the phenylalanine deaminase of Hordeum vulgare". The Journal of Biological Chemistry. 236 (10): 2692–8. doi: 10.1016/S0021-9258(19)61721-7 . PMID   14458851.
  12. ↑ Alunni S, Cipiciani A, Fioroni G, Ottavi L (April 2003). "Mechanisms of inhibition of phenylalanine ammonia-lyase by phenol inhibitors and phenol/glycine synergistic inhibitors". Archives of Biochemistry and Biophysics. 412 (2): 170–5. doi:10.1016/s0003-9861(03)00007-9. PMID   12667480.
  13. 1 2 Langer B, Langer M, Rétey J (2001). "Methylidene-imidazolone (MIO) from histidine and phenylalanine ammonia-lyase". Advances in Protein Chemistry. 58: 175–214. doi:10.1016/s0065-3233(01)58005-5. ISBN   9780120342587. PMID   11665488.
  14. ↑ Frey PA, Hegeman AD (2007). "Methylene imidazolone-dependent elimination and addition: phenylalanine ammonia-lyase". Enzymatic Reaction Mechanisms . Oxford University Press. pp.  460–466. ISBN   9780195352740.
  15. 1 2 Ritter H, Schulz GE (December 2004). "Structural basis for the entrance into the phenylpropanoid metabolism catalyzed by phenylalanine ammonia-lyase". The Plant Cell. 16 (12): 3426–36. doi:10.1105/tpc.104.025288. PMC   535883 . PMID   15548745.
  16. ↑ Rétey, János (2003). "Discovery and role of methylidene imidazolone, a highly electrophilic prosthetic group". Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics. 1647 (1–2): 179–184. doi:10.1016/S1570-9639(03)00091-8. PMID   12686130.
  17. 1 2 3 Calabrese JC, Jordan DB, Boodhoo A, Sariaslani S, Vannelli T (September 2004). "Crystal structure of phenylalanine ammonia lyase: multiple helix dipoles implicated in catalysis". Biochemistry. 43 (36): 11403–16. doi:10.1021/bi049053+. PMID   15350127.
  18. ↑ Sato T, Kiuchi F, Sankawa U (1 January 1982). "Inhibition of phenylalanine ammonia-lyase by cinnamic acid derivatives and related compounds". Phytochemistry. 21 (4): 845–850. doi:10.1016/0031-9422(82)80077-0.
  19. ↑ Pilbák S, Tomin A, Rétey J, Poppe L (March 2006). "The essential tyrosine-containing loop conformation and the role of the C-terminal multi-helix region in eukaryotic phenylalanine ammonia-lyases". The FEBS Journal. 273 (5): 1004–19. doi: 10.1111/j.1742-4658.2006.05127.x . PMID   16478474. S2CID   33002042.
  20. ↑ Powers M (May 29, 2018). "Biomarin aces final exam: Palynziq gains FDA approval to treat PKU in adults". BioWorld.
  21. ↑ Levy HL, Sarkissian CN, Stevens RC, Scriver CR (June 2018). "Phenylalanine ammonia lyase (PAL): From discovery to enzyme substitution therapy for phenylketonuria". Molecular Genetics and Metabolism. 124 (4): 223–229. doi:10.1016/j.ymgme.2018.06.002. PMID   29941359. S2CID   49411168.
  22. ↑ Urban, L.; Chabane Sari, D.; Orsal, B.; Lopes, M.; Miranda, R.; Aarrouf, J. (2018). "UV-C light and pulsed light as alternatives to chemical and biological elicitors for stimulating plant natural defenses against fungal diseases". Scientia Horticulturae . 235. Elsevier: 452–459. doi:10.1016/j.scienta.2018.02.057. ISSN   0304-4238. S2CID   90436989.
  23. ↑ Straathof AJ, Adlercreutz P (2014). Applied Biocatalysis. CRC Press. p. 146. ISBN   9781482298420.
  24. ↑ Hughes A (2009). Amino Acids, Peptides and Proteins in Organic Chemistry Volume 1. Weinheim Germany: Wiley VCH. p. 94. ISBN   9783527320967.
  25. ↑ Renard G, Guilleux J, Bore C, Malta-Valette V, Lerner D (1992). "Synthesis of L-phenylalanine analogs by Rhodotorula glutinis. Bioconversion of cinnamic acids derivatives". Biotechnology Letters. 14 (8): 673–678. doi:10.1007/BF01021641. S2CID   46423586.
  26. ↑ Lovelock SL, Turner NJ (October 2014). "Bacterial Anabaena variabilis phenylalanine ammonia lyase: a biocatalyst with broad substrate specificity". Bioorganic & Medicinal Chemistry. 22 (20): 5555–7. doi:10.1016/j.bmc.2014.06.035. PMID   25037641.
  27. ↑ Showa D, Hirobumi A. "Production process of L-phenylalanine derivatives by microorganisms". Google Patents. Hirobumi Central Research Laboratory. Retrieved 20 July 2014.

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