Purification and characterization of phenylalanine ammonia-lyase from Ustilago maydis

Phytochemistry (Impact Factor: 3.05). 01/1996; 43(2):351-357. DOI: 10.1016/0031-9422(96)00282-8

ABSTRACT Phenylalanine ammonia-lyase (PAL; EC. has been purified to homogeneity from liquid-cultured cells of the phytopathogenic fungus Ustilago maydis by use of heat treatment, protamine and ammonium sulphate precipitation, ion-exchange and gel filtration chromatography, and preparative PAGE. Its native molecular mass was estimated as 320±20 kDa and its subunit molecular mass as 80 kDa. No isoforms of the enzyme were detected, and there was no evidence of glycosylation of the protein. Ustilago PAL was most active at pH 8.8–9.2 and 30° and had a Km for l-phenylalanine of 1.05 mM. The enzyme did not deaminate l-tyrosine. The synthetic inhibitor 2-aminoindan-2-phosphonic acid (AIP) strongly inhibited the enzyme, as did sulphhydryl reagents and carbonyl reagents, whereas t-cinnamate was only moderately inhibitory. Ustilago PAL activity had no requirement for metal ion cofactors, but was inhibited by heavy metal ions (Ag+, Cu2+, and Hg2+). Polyclonal antibodies raised against the purified enzyme readily recognized U. maydis PAL in solution and on Western blots, but only weakly cross-reacted with higher plant PAL.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Phenylalanine ammonia-lyase (PAL) catalyzes the first reaction in phenylpropanoid pathway leading to the production of phenolic compounds with a wide range of biological functions. The cDNA encoding PAL was isolated from Phyllostachys edulis by reverse transcription-polymerase chain reaction (RT-PCR) and by 5' and 3' rapid amplification of cDNA ends, and was designated as PePAL. The full length of PePAL was 2,503 bp which contained an open reading frame (ORF) encoding a peptide of 701 amino acids, with a theoretic isoelectric point of 6.49 and a calculated molecular mass of 75.7 kDa. PePAL was heterologously expressed in Escherichia coli and the recombinant proteins exhibited both PAL and tyrosine ammonia-lyase (TAL) activities. The optimum temperature and pH of the recombinant PePAL were 50 °C and 8.5-9.0, respectively. The K (m) and V (max) values for L-phenylalanine was calculated as 422 μM and 45.9 nM s⁻¹, while for L-tyrosine were 78 μM and 7.09 nM s⁻¹, respectively. Tissue-specific expression assay showed that PePAL expressed highest in stem and sheath, followed by leaf, and lowest in root. Though the accumulation of PePAL proteins was observed in all these four organs by Western blotting, the highest was detected in leaf. Immunohistochemistry study showed that PePAL was localized primarily in vascular bundles and part of sclerenchyma cells of leaf, sheath and root. These results suggested that PePAL had similar expression pattern and biochemical properties with PALs in other plants, which laid the basis for molecular engineering to improve the quality of bamboo products. KEY MESSAGE: PePAL was a protein with bifunctional enzyme activities of PAL and TAL as shown in vitro assays, and localized primarily in bamboo vascular bundles.
    Plant Cell Reports 05/2012; 31(7):1345-56. · 2.51 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Phenylalanine ammonia-lyase (PAL) is the first key enzyme of the phenypropanoid pathway. A full-length cDNA of PAL gene was isolated from Juglans regia for the first time, and designated as JrPAL. The full-length cDNA of the JrPAL gene contained a 1935bp open reading frame encoding a 645-amino-acid protein with a calculated molecular weight of about 70.4 kD and isoelectric point (pI) of 6.7. The deduced JrPAL protein showed high identities with other plant PALs. Molecular modeling of JrPAL showed that the 3D model of JrPAL was similar to that of PAL protein from Petroselinum crispum (PcPAL), implying that JrPAL may have similar functions with PcPAL. Phylogenetic tree analysis revealed that JrPAL shared the same evolutionary ancestor of other PALs and had a closer relationship with other angiosperm species. Transcription analysis revealed that JrPAL was expressed in all tested tissues including roots, stems, and leaves, with the highest transcription level being found in roots. Expression profiling analyses by real-time PCR revealed that JrPAL expression was induced by a variety of abiotic and biotic stresses, including UV-B, wounding, cold, abscisic acid and salicylic acid.
    Molecules 01/2012; 17(7):7810-23. · 2.43 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: L-Phenylalanine is one of the essential amino acids that cannot be synthesized in mammals in adequate amounts to meet the requirements for protein synthesis. Fungi and plants are able to synthesize phenylalanine via the shikimic acid pathway. L-Phenylalanine, derived from the shikimic acid pathway, is used directly for protein synthesis in plants or metabolized through the phenylpropanoid pathway. This phenylpropanoid metabolism leads to the biosynthesis of a wide array of phenylpropanoid secondary products. The first step in this metabolic sequence involves the action of phenylalanine ammonia-lyase (PAL). The discovery of PAL enzyme in fungi and the detection of (14)CO(2) production from (14)C-ring-labeled phenylalanine and cinnamic acid demonstrated that certain fungi can degrade phenylalanine by a pathway involving an initial deamination to cinnamic acid, as happens in plants. In this review, we provide background information on PAL and a recent update on the presence of PAL genes in fungi.
    Mycobiology 12/2011; 39(4):257-65.