Article

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

Department of Microbiology and Immunology, University of British Columbia - Vancouver, Vancouver, British Columbia, Canada
Phytochemistry (Impact Factor: 2.55). 09/1996; 43(2):351-357. 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.

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    • "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. "
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    ABSTRACT: Export Date: 18 October 2014
    Full-text · Article · Jan 2014 · Critical Reviews in Biotechnology
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    • "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. "
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    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
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    • "Most reported PALs range in size from 300 to 340 kDa in native molecular mass. Some examples of exceptions are reported masses of 152 kDa in Ocimum basilicum [25], 226 kDa in the bacterium, Streptomyces [11], 250 kDa in Helianthus annuus [5], 266 kDa in Fragaria ananassa [20], 320 kDa in Ustilago maydis [26], and 560 kDa in Alternaria [21]. PAL is normally a homo-tetrameric protein consisting of four identical subunits. "
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    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.
    Full-text · Article · Dec 2011 · Mycobiology
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