Purification and characterization of phenylalanine ammonia-lyase from Ustilago maydis. Phytochemistry

ArticleinPhytochemistry 43(2):351-357 · September 1996with13 Reads
Impact Factor: 2.55 · DOI: 10.1016/0031-9422(96)00282-8
Abstract

Phenylalanine ammonia-lyase (PAL; EC. 4.3.1.5) 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.

    • "About 80% recovery yield and 32.6-fold purification factor of PAL were obtained (Monge et al., 1995). PAL has also been purified to homogeneity from liquid-cultured cells of the phytopathogenic fungus U. maydis by use of heat treatment, protamine and ammonium sulfate precipitation, ion-exchange and gel filtration chromatography (Kim et al., 1996). In addition, most PALs are considered to be hydrophobic proteins. "
    [Show abstract] [Hide abstract] ABSTRACT: Export Date: 18 October 2014
    Full-text · Article · Jan 2014 · Critical Reviews in Biotechnology
    J.D. Cui J.D. Cui J.Q. Qiu J.Q. Qiu X.W. Fan X.W. Fan +1 more author... S.R. Jia S.R. Jia
    0Comments 7Citations
    • "About 80% recovery yield and 32.6-fold purification factor of PAL were obtained (Monge et al., 1995). PAL has also been purified to homogeneity from liquid-cultured cells of the phytopathogenic fungus U. maydis by use of heat treatment, protamine and ammonium sulfate precipitation, ion-exchange and gel filtration chromatography (Kim et al., 1996). In addition, most PALs are considered to be hydrophobic proteins. "
    [Show abstract] [Hide abstract] ABSTRACT: Abstract Phenylalanine ammonia lyase (PAL) catalyzes the nonoxidative deamination of L-phenylalanine to form trans-cinnamic acid and a free ammonium ion. It plays a major role in the catabolism of L-phenylalanine. The presence of PAL has been reported in diverse plants, some fungi, Streptomyces and few Cyanobacteria. In the past two decades, PAL has gained considerable significance in several clinical, industrial and biotechnological applications. Since its discovery, much knowledge has been gathered with reference to the enzyme's importance in phenyl propanoid pathway of plants. In contrast, there is little knowledge about microbial PAL. Furthermore, the commercial source of the enzyme has been mainly obtained from the fungi. This study focuses on the recent advances on the physiological role of microbial PAL and the improvements of PAL biotechnological production both from our laboratory and many others as well as the latest advances on the new applications of microbial PAL.
    Full-text · Article · May 2013 · Critical Reviews in Biotechnology
    0Comments 9Citations
    • "Thus, the study on the characterization and expression pattern of the genes involved in the flavonoid biosynthetic pathway, in particular the well-documented genes such as PAL, is essential to further understanding the mechanism of stress resistance and the biosynthesis of flavonoids [14]. PAL is present in all higher plants studied and is also found in some fungi [15,16], cyanobacteria [17], and Streptomyces [18]. Because of its crucial role in the biosynthesis of flavonoids, lignins, and phytoalexins and stress responses, PAL and its gene are widely studied [3,19]. "
    [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.
    Full-text · Article · Dec 2012 · Molecules
    0Comments 12Citations
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