Phenylalanine ammonia-lyase (PAL) is the key entry enzyme of plant phenylpropanoid pathway. It plays an important role in the biosynthesis of podophyllotoxin, an anti-tumor lignan that is currently produced from its main natural source Sinopodophyllum hexandrum (Royle) Ying. In this study, we cloned the gene ShPAL encoding phenylalanine ammonia-lyase by RT-PCR from the root of S. hexandrum ecotype inhabited in the Aba' district, Sichuan, based on its public SRA transcriptome data-package. Bioinformatics analyses showed that the ShPAL-encoded protein is composed of 711 amino acids, contains the conserved domains of PAL, and has the signature motif within the active center of aromatic ammonia-lyases. Moreover, ShPAL protein was predicted to have a secondary structure mainly composed of α-helix and random coil, a typical 'seahorse' shape monomer tertiary structure, and a homologous tetramer three-dimensional structure by Swiss-Modelling. The phylogenetic lineage analysis indicated ShPAL was of the highest sequence identity and the shortest evolutionary distance with the PAL of Epimedium sagittatum from the same Berberidaceae family. Subcellular localization experiments showed that ShPAL protein was mainly distributed in the cytoplasm, despite of a minority on the endoplasmic reticulum membrane. Furthermore, ShPAL protein was recombinantly expressed in Escherichia coli and purified by histidine-tag affinity chromatography. Its enzymatic activity was determined up to 20.91 U/mg, with the optimum temperature of 41 ℃ and pH of 9.0. In contrast, the enzyme activity of its F130H mutant decreased by about 23.6%, yet with the same trends of change with temperature and pH, confirming that phenylalanine at this position does affect the substrate specificity of PAL. Both the wild type and the mutant have relatively poor thermostability, but good pH-stability. These results may help to further investigate the regulatory role of PAL in the process of podophyllotoxin biosynthesis and advance the heterologous synthesis of podophyllotoxin to protect the germplasm resource of S. hexandrum. They also demonstrate that ShPAL has a potential application in biochemical industry and biomedicine.
Phenylalanine Ammonia-Lyase/metabolism* ; Podophyllotoxin ; Phylogeny ; Cloning, Molecular

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 14CO2 production from 14C-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.
Amino Acids, Essential ; Cinnamates ; Deamination ; Fungi ; Mammals ; Phenylalanine ; Phenylalanine Ammonia-Lyase ; Plants ; Resin Cements ; Shikimic Acid

Phenylalanine ammonia-lyase (PAL), which catalyzes the conversion from L-phenylalanine to trans-cinnamic acid, is a well-known key enzyme and a connecting step between primary and secondary metabolisms in the phenylpropanoid biosynthetic pathway of plants and microbes. Schisandra chinensis, a woody vine plant belonging to the family of Magnoliaceae, is a rich source of dibenzocyclooctadiene lignans exhibiting potent activity. However, the functional role of PAL in the biosynthesis of lignan is relatively limited, compared with those in lignin and flavonoids biosynthesis. Therefore, it is essential to clone and characterize the PAL genes from this valuable medicinal plant. In this study, molecular cloning and characterization of three PAL genes (ScPAL1-3) from S. chinensis was carried out. ScPALs were cloned using RACE PCR. The sequence analysis of the three ScPALs was carried out to give basic characteristics followed by docking analysis. In order to determine their catalytic activity, recombinant protein was obtained by heterologous expression in pCold-TF vector in Escherichia coli (BL21-DE3), followed by Ni-affinity purification. The catalytic product of the purified recombinant proteins was verified using RP-HPLC through comparing with standard compounds. The optimal temperature, pH value and effects of different metal ions were determined. V_max, K_cat and K_m values were determined under the optimal conditions. The expression of three ScPALs in different tissues was also determined. Our work provided essential information for the function of ScPALs.
Cloning, Molecular ; Escherichia coli/metabolism* ; Phenylalanine/metabolism* ; Phenylalanine Ammonia-Lyase/chemistry* ; Recombinant Proteins ; Schisandra/genetics*

Animals ; Biopterin ; analogs & derivatives ; therapeutic use ; Genetic Therapy ; Humans ; Phenylalanine Ammonia-Lyase ; administration & dosage ; Phenylketonurias ; therapy

In order to reveal the induced resistance mechanism of tobacco treated with copper solution to potato virus Y-vein necrosis strain (PVY(N)), disease indexes, contents of virus and some physiological and biochemical indexes in tobacco were studied. The results showed that when treated at the copper concentration of 0.8 mg x L(-1), the symptom displayed and vein necrosis on tobacco were postponed, the disease index and content of virus sharply decreased , and the content of chlorophyll a, chlorophyll b and phenylalanine ammonia lyase (PAL) activity remarkably increased. Furthermore, vein necrosis closely linked to contents of total phenol and flavonoid. In this study, the contents of total phenol and flavonoid were promoted when treated with a solution at the copper concentration of 0.8 mg x L(-1). But the contents of total phenol and flavonoid reached to the first peak at the 3rd day after inoculation, and then decreased to the lowest levels which even were lower than those of the control after inoculating PVY(N). Then the contents of total phenol and flavonoid increased slowly from the 6td but still lower than those of the control. The result implied that spraying copper solution might play an important role in induced resistance of tobacco to vein necrosis disease and strengthen the antiviral capability to PVY(N).
Chlorophyll ; metabolism ; Copper ; pharmacology ; Immunity, Innate ; drug effects ; Phenylalanine Ammonia-Lyase ; metabolism ; Potyvirus ; growth & development ; Tobacco ; drug effects ; metabolism ; virology

Phenylalanine ammonia-lyase (PAL) gene is known to be expressed in plants, and is involved in the differentiation, growth and synthesis of secondary metabolites. However, its expression in fungi remains to be explored. To understand its expression in mushroom fungi, the PAL gene of the edible mushroom Flammulina velutipes (Fvpal) was cloned and characterized. The cloned Fvpal consists of 2,175 bp, coding for a polypeptide containing 724 amino acids and having 11 introns. The translated amino acid sequence of Fvpal shares a high identity (66%) with that of ectomycorrhizal fungus Tricholoma matsutake. Distinctively, the Fvpal expression in the mycelium was higher in minimal medium supplemented with L-tyrosine than with other aromatic amino acids. During cultivation of the mushroom on sawdust medium, Fvpal expression in the fruit body correspondingly increased as the mushroom grew. In the fruiting body, Fvpal was expressed more in the stipe than in the pileus. These results suggest that F. velutipes PAL activity differs in the different organs of the mushroom. Overall, this is first report to show that the PAL gene expression is associated with mushroom growth in fungi.
Agaricales* ; Amino Acid Sequence ; Amino Acids ; Amino Acids, Aromatic ; Clinical Coding ; Clone Cells* ; Cloning, Organism* ; Flammulina* ; Fruit* ; Fungi ; Gene Expression ; Introns ; Mycelium* ; Phenylalanine Ammonia-Lyase* ; Phenylalanine* ; Tricholoma ; Tyrosine

Rosmarinic acid (RA), a phenolic acid, is one of the important secondary metabolites produced in Salvia miltiorrhiza. To observe the influence of salicylic acid (SA), an elicitor, on the synthesis of RA and related enzymes, we treated the cell suspension cultures of S. miltiorrhiza with SA and L-a-aminooxy-beta-phenylpropionic acid (AOPP), a competitive inhibitor of tyrosine aminotransferase (TAT). Under this condition, the activities of related enzymes, such as phenylalanine ammonia-lyase and TAT were traced and assayed; the accumulative amount of RA was measured. The results showed that the PAL activity reached the peak at 4 h, 124% higher than that of the control, and the content of RA reached its maximum ((5.914 +/- 0.296) mg/g dry weight) at 8 h, after treated by 6.25 mg/L SA on day 6 of the suspension culture. The results of treatment with 0.1 micromol/L AOPP showed that AOPP affected little on the TAT activity, while the PAL activity was significantly influenced, with 44% lower than that of the control at 6 h. Meanwhile, the reduced accumulation of RA ((4.709 +/- 0.204) mg/g dry weight) paralleled with the decrease in PAL activity. The co-treatment by 0.1 micromol/L AOPP and 6.25 mg/L SA relieved the restriction imposed by AOPP on PAL, and made the cell cultures accumulate more RA than sole treatment with AOPP, indicated that SA induced the accumulation of RA in suspension cell culture of S. miltiorrhiza, and the rate-limiting effect of PAL was stronger than TAT.
Cell Culture Techniques ; methods ; Cinnamates ; metabolism ; Depsides ; metabolism ; Phenylalanine Ammonia-Lyase ; metabolism ; Plant Cells ; metabolism ; Salicylic Acid ; pharmacology ; Salvia miltiorrhiza ; cytology ; growth & development ; metabolism ; Suspensions ; Tyrosine Transaminase ; metabolism

Nitric oxide has emerged as a key signaling molecule in plants recently. The role of nitric oxide in elicitor-induced defense responses of plants has been extensively investigated. In this work, sodium nitroprusside was utilized as the donor of nitric oxide to investigate the effects of exogenous nitric oxide on hypericin production and cell growth of suspension cell cultures of Hypericum perforatum L.. Compared with the untreated Hypericum perforatum L. suspension cells, external application of 0.5 and 15.0 mmol/L sodium nitroprusside induced 1.4 and 0.5-fold dry cell weight, and 0.9 and 2.1-fold hypericin content respectively. The results showed that low concentration of sodium nitroprusside promoted the growth of Hypericum perforatum L. suspension cells, while high concentration of sodium nitroprusside enhanced hypericin biosynthesis in Hypericum perforatum L. suspension cells. The maximum hypericin production was achieved by adding 0.5 mmol/L and 15.0 mmol/L sodium nitroprusside to the culture at day 0 and day 14 respectively, increasing the total hypericin yield by nearly 3.2-fold. The effects of sodium nitroprusside on hypericin content and growth of Hypericum perforatum L. suspension cells were abolished by nitric oxide specific scavenger 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, which indicated that the effects of the application of sodium nitroprusside were caused by nitric oxide released from sodium nitroprusside rather than sodium nitroprusside itself. The results also showed that 15.0 mmol/L sodium nitroprusside stimulated the activities of phenylalanine ammonia-lyase (PAL), one of the key enzymes of phenylpropanoid pathway, in suspension cells of Hypericum perforatum L., which suggested that the synthetic pathway of hypericin might be activated by NO through triggering the defense responses of Hypericum perforatum L. suspension cells.
Cells, Cultured ; Hypericum ; cytology ; drug effects ; metabolism ; Nitric Oxide ; metabolism ; Nitroprusside ; pharmacology ; Perylene ; analogs & derivatives ; metabolism ; Phenylalanine Ammonia-Lyase ; metabolism ; Plant Growth Regulators ; biosynthesis

To construct a new high effective genetic engineering strain which can express active PAL enzyme in Lactococcus lactis (L.L), and acquire better effect on curing hyperphenylalaninemia rats, Firstly translational fusion vector and transcriptional fusion vector were constructed in E. coli MC1061, and then PAL cDNA was transformed into L.L. Two kinds of high effect strain were compared with their enzyme activity and animal experiment was carried out. The results showed: (1) Two kinds of engineering L.L. were obtained and translational fusion strain has higher level enzyme activity. (2) The amount of transcinnamic aicd reach peak when induced for 6 hours. (3) The blood phe level of the treated rats was significantly reduced compared with non-treated rats when receiving fresh p(NZ8048-PAL)1/NZ9000. The engineering L.L(translational fusion strain) can significantly reduce the blood phe level of the hyperphenylalaninemia rats, which has more superiority than pMG36e-PAL/L. L.
Animals ; Escherichia coli ; genetics ; Genetic Therapy ; Lactococcus lactis ; genetics ; Male ; Phenylalanine Ammonia-Lyase ; genetics ; Phenylketonurias ; therapy ; Polymerase Chain Reaction ; Rats ; Rats, Wistar

OBJECTIVETo study the effect of endophytic fungi.

METHODA. formosanus, harvested after having been cultured for age, statistics were taken, fresh weight and dry weight were gained, and enzyme activities of chitinase, beta-1,3-glucase, phenylalanine ammonia-lyase and polyphenoloxidase were determined.

RESULTThe survial rates of A. formosanus inoculated with endophytic fungi was 100%. The effect of fungi on fresh weight was very significant (P < 0.01). The effect of fungi on dry weight was significant (P > 0.05). The four enzyme activities were enhanced by endophytic fungi, comparision with the controls.

CONCLUSIONSurvial rates of A. formosanus can be increased by using endophytic fungi in vitro culture.

Catechol Oxidase ; analysis ; Chitinases ; analysis ; Mycorrhizae ; physiology ; Orchidaceae ; enzymology ; growth & development ; microbiology ; Phenylalanine Ammonia-Lyase ; analysis ; Plants, Medicinal ; enzymology ; growth & development ; microbiology ; Symbiosis ; beta-Glucans ; analysis