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

Abstract

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...

Grass roots chemistry: meta -Tyrosine, an herbicidal nonprotein amino acid

“…However, we did not observe significant changes in the abundance of aromatic amino acids in Arabidopsis seedlings growing on growth-inhibiting levels of m-tyrosine. The majority of phenylalanine flux in plants passes through phenylalanine ammonia lyase, and some plant phenylalanine ammonia lyase enzymes can convert m-tyrosine to m-hydroxycinnamate (22). However, m-hydroxycinnamate concentrations up to 80 M did not inhibit root growth (data not shown), indicating that enzymatic conversion by phenylalanine ammonia lyase probably does not contribute to m-tyrosine toxicity.…”

Structural Basis for the Entrance into the Phenylpropanoid Metabolism Catalyzed by Phenylalanine Ammonia-Lyase

“…Because of inefficient codon usage, heterologous expression of the 2.2-kb gene of isoenzyme-1 of the PAL from parsley (Petroselinum crispum) in Escherichia coli failed to yield enough homogenous protein for a structure analysis (Schuster and Retey, 1995). Therefore, we used a gene especially designed for E. coli (Baedeker and Schulz, 1999).…”

Possibility of Bacterial Recruitment of Plant Genes Associated with the Biosynthesis of Secondary Metabolites

“…All three enzymes have the unique prosthetic group 4-methylidene imidazol-5-one (Schwede et al, 1999;Rother et al, 2001), indicating a very similar catalytic mechanism. However, because PAL is the first enzyme in plant phenylpropanoid secondary metabolism, it is the best studied member of this enzyme family (Schuster and Retey, 1995).…”