Serine‐202 is the putative precursor of the active site dehydroalanine of phenylalanine ammonia lyase Site‐directed mutagenesis studies on the enzyme from parsley (<i>Petroselinum crispum</i> L.)

Schuster, B; Rétey, János

doi:10.1016/0014-5793(94)00681-4

Cited by 51 publications

(41 citation statements)

References 36 publications

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“…These procedures for recombinant wild-type PAL and the mutant S202A have been described in detail (13). The PAL mutant S202T was constructed and overexpressed by an analogous method using the pT7.7 expression system.…”

Section: Resultsmentioning

confidence: 99%

“…Site-directed mutagenesis was performed by following the protocol of the Amersham mutagenesis kit (Sculptor). PALMutS202A and PALMutS202T were constructed as described by Schuster and Retey (13). The oligonucleotide sequence used in this mutagenesis reaction was 5'-CACTGCTACTGGTGATC-3'.…”

Section: Methodsmentioning

confidence: 99%

“…Heterologous expression allowed the production of mutants by site-directed mutagenesis. The PAL mutant S202A and the HAL mutants S143A and S143T were virtually inactive with L-phenylalanine and L-histidine as substrates, respectively (9,10,13,14). Surprisingly, L-5-nitrohistidine, known to be a moderately good substrate of HAL, reacted with the dehydroalanine-less mutants S143A and S143T and with wild-type HAL to the same extent.…”

Section: Introductionmentioning

confidence: 95%

“…Such a reaction should enhance the leaving ability of the amino group. Recently, this laboratory has identified the precursor of the dehydroalanine in HAL from Pseudomonas putida and PAL from parsley to be Ser-143 and Ser-202, respectively (9,10,13). Heterologous expression allowed the production of mutants by site-directed mutagenesis.…”

mentioning

confidence: 99%

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The mechanism of action of phenylalanine ammonia-lyase: the role of prosthetic dehydroalanine.

Schuster

Rétey

1995

Proc. Natl. Acad. Sci. U.S.A.

127

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Phenylalanine ammonia-lyase (EC 4.3.1.5) from parsley is posttranslationally modified by dehydrating its Ser-202 to the catalytically essential dehydroalanine prosthetic group. The codon of Ser-202 was changed to those of alanine and threonine by site-directed mutagenesis. These mutants and the recombinant wild-type enzyme, after treatment with sodium borohydride, were virtually inactive with L-phenylalanine as substrate but catalyzed the deamination of L-4-nitrophenylalanine, which is also a substrate for the wild-type enzyme. Although the mutants reacted about 20 times slower with L-4-nitrophenylalanine than the wild-type enzyme, their V... for L-4-nitrophenylalanine was two orders of magnitude higher than for L-phenylalanine. In contrast to L-tyrosine, which was a poor substrate, DL-3-hydroxyphenylalanine (DL-m-tyrosine) was converted by phenylalanine ammonia-lyase at a rate comparable to that of L-phenylalanine. These results suggest a mechanism in which the crucial step is an electrophilic attack of the prosthetic group at position 2 or 6 of the phenyl group. In the resulting carbenium ion, the 18-Hsi atom is activated in a similar way as it is in the nitro analogue. Subsequent elimination of ammonia, concomitant with restoration of both the aromatic ring and the prosthetic group, completes the catalytic cycle.Phenylalanine ammonia-lyase (PAL) is an important plant enzyme that converts L-phenylalanine into trans-cinnamic acid, which in turn is the precursor of various phenylpropanoids, such as lignins, flavonoids, and coumarins (1, 2). It has been known for a long time that PAL contains a catalytically essential dehydroalanine residue (3). Although the latter occurs in a number of natural peptides (4, 5) and has been postulated as an intermediate in the conversion of L-serine residues into D-alanine (6), it is shared only by one related enzyme, histidine ammonia-lyase (HAL) (7-10).The electrophilic property of the prosthetic dehydroalanine has been demonstrated by addition of various nucleophiles, such as nitromethane (7-11), cyanide (12), and sodium borohydride (3,8). All these reagents caused inactivation and, using radiolabel, incorporation of the radioisotope into the expected product. Twenty-five years ago a mechanism was proposed for the PAL and HAL reactions involving as an initial step the nucleophilic attack at the dehydroalanine residue by the amino group of the substrate (3, 7). Such a reaction should enhance the leaving ability of the amino group. Recently, this laboratory has identified the precursor of the dehydroalanine in HAL from Pseudomonas putida and PAL from parsley to be Ser-143 and Ser-202, respectively (9, 10, 13). Heterologous expression allowed the production of mutants by site-directed mutagenesis. The PAL mutant S202A and the HAL mutants S143A and S143T were virtually inactive with L-phenylalanine and L-histidine as substrates, respectively (9,10,13,14). Surprisingly, L-5-nitrohistidine, known to be a moderately good substrate of HAL, reacted with the dehydroalanine-less m...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 95%

mentioning

confidence: 99%

See 2 more Smart Citations

The mechanism of action of phenylalanine ammonia-lyase: the role of prosthetic dehydroalanine.

Schuster

Rétey

1995

Proc. Natl. Acad. Sci. U.S.A.

127

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“…PvPAL was found to have a conserved motif (GTITASGDLVPLSYIA) corresponding to the active site of PAL. 22) Phylogenetic analysis of these sequences further clarified their evolutionary relationships. As shown in Supplemental information Fig.…”

Section: Fig 3 Multiple Alignments Of the Amino Acid Sequence Of Pvmentioning

confidence: 99%

Molecular Cloning and Characterization of Genes Involved in Rosmarinic Acid Biosynthesis from <i>Prunella vulgaris</i>

Kim

Shin

Tuấn

et al. 2014

Biological & Pharmaceutical Bulletin

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Prunella vulgaris L., commonly known as "self-heal" or "heal-all," is a perennial herb with a long history of medicinal use. Phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumarate:coenzyme-A (CoA) ligase (4CL) are important enzymes in the phenylpropanoid pathway and in the accumulation of rosmarinic acid (RA), which is a major secondary metabolite in P. vulgaris. In this study, we isolated cDNAs encoding PvPAL, PvC4H, and Pv4CL from P. vulgaris using rapid amplification of cDNA ends polymerase chain reaction (PCR). The amino acid sequence alignments of PvPAL, PvC4H, and Pv4CL showed high sequence identity to those of other plants. Quantitative real-time PCR analysis was used to determine the transcript levels of genes involved in RA biosynthesis in the flowers, leaves, stems, and roots of P. vulgaris. The transcript levels of PvPAL, PvC4H, and Pv4CL1 were the highest in flowers, whereas Pv4CL2 was the highest in roots. High-performance liquid chromatography analysis also showed the highest RA content in the flowers (3.71 mg/g dry weight). We suggest that the expression of the PvPAL, PvC4H, and Pv4CL1 genes is correlated with the accumulation of RA. Our results revealed that P. vulgaris flowers are appropriate for medicinal usage, and our findings provide support for increasing RA production in this plant.

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Spektroskopischer Nachweis eines 4‐Methylidenimidazol‐5‐ons sowohl in Histidin‐ als auch in Phenylalanin‐Ammoniak‐Lyasen

Röther¹,

Merkel²,

Rétey³

2000

Angewandte Chemie

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Serine‐202 is the putative precursor of the active site dehydroalanine of phenylalanine ammonia lyase Site‐directed mutagenesis studies on the enzyme from parsley (Petroselinum crispum L.)

Cited by 51 publications

References 36 publications

The mechanism of action of phenylalanine ammonia-lyase: the role of prosthetic dehydroalanine.

The mechanism of action of phenylalanine ammonia-lyase: the role of prosthetic dehydroalanine.

Molecular Cloning and Characterization of Genes Involved in Rosmarinic Acid Biosynthesis from <i>Prunella vulgaris</i>

Spektroskopischer Nachweis eines 4‐Methylidenimidazol‐5‐ons sowohl in Histidin‐ als auch in Phenylalanin‐Ammoniak‐Lyasen

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