A different approach to treatment of phenylketonuria: phenylalanine degradation with recombinant phenylalanine ammonia lyase.
Phenylketonuria (PKU), with its associated hyperphenylalaninemia (HPA) and mental retardation, is a classic genetic disease and the first to have an identified chemical cause of impaired cognitive development. Treatment from birth with a low phenylalanine diet largely prevents the deviant cognitive phenotype by ameliorating HPA and is recognized as one of the first effective treatments of a genetic disease. However, compliance with dietary treatment is difficult and when it is for life, as now recommended by an internationally used set of guidelines, is probably unrealistic. Herein we describe experiments on a mouse model using another modality for treatment of PKU compatible with better compliance using ancillary phenylalanine ammonia lyase (PAL, EC 22.214.171.124) to degrade phenylalanine, the harmful nutrient in PKU; in this treatment, PAL acts as a substitute for the enzyme phenylalanine monooxygenase (EC 126.96.36.199), which is deficient in PKU. PAL, a robust enzyme without need for a cofactor, converts phenylalanine to trans-cinnamic acid, a harmless metabolite. We describe (i) an efficient recombinant approach to produce PAL enzyme, (ii) testing of PAL in orthologous N-ethyl-N'-nitrosourea (ENU) mutant mouse strains with HPA, and (iii) proofs of principle (PAL reduces HPA)-both pharmacologic (with a clear dose-response effect vs. HPA after PAL injection) and physiologic (protected enteral PAL is significantly effective vs. HPA). These findings open another way to facilitate treatment of this classic genetic disease. (+info)
Sugar- and nitrogen-dependent regulation of an Amanita muscaria phenylalanine ammonium lyase gene.
The cDNA of a key enzyme of secondary metabolism, phenylalanine ammonium lyase, was identified for an ectomycorrhizal fungus by differential screening of a mycorrhizal library. The gene was highly expressed in hyphae grown at low external monosaccharide concentrations, but its expression was 30-fold reduced at elevated concentrations. Gene repression was regulated by hexokinase. (+info)
Inverse relationship between systemic resistance of plants to microorganisms and to insect herbivory.
Pre-inoculation of plants with a pathogen that induces necrosis leads to the development of systemic acquired resistance (SAR) to subsequent pathogen attack . The phenylpropanoid-derived compound salicylic acid (SA) is necessary for the full expression of both local resistance and SAR  . A separate signaling pathway involving jasmonic acid (JA) is involved in systemic responses to wounding and insect herbivory  . There is evidence both supporting and opposing the idea of cross-protection against microbial pathogens and insect herbivores  . This is a controversial area because pharmacological experiments point to negative cross-talk between responses to systemic pathogens and responses to wounding   , although this has not been demonstrated functionally in vivo. Here, we report that reducing phenylpropanoid biosynthesis by silencing the expression of phenylalanine ammonialyase (PAL) reduces SAR to tobacco mosaic virus (TMV), whereas overexpression of PAL enhances SAR. Tobacco plants with reduced SAR exhibited more effective grazing-induced systemic resistance to larvae of Heliothis virescens, but larval resistance was reduced in plants with elevated phenylpropanoid levels. Furthermore, genetic modification of components involved in phenylpropanoid synthesis revealed an inverse relationship between SA and JA levels. These results demonstrate phenylpropanoid-mediated cross-talk in vivo between microbially induced and herbivore-induced pathways of systemic resistance. (+info)
Hydrogen peroxide from the oxidative burst is neither necessary nor sufficient for hypersensitive cell death induction, phenylalanine ammonia lyase stimulation, salicylic acid accumulation, or scopoletin consumption in cultured tobacco cells treated with elicitin.
H(2)O(2) from the oxidative burst, cell death, and defense responses such as the production of phenylalanine ammonia lyase (PAL), salicylic acid (SA), and scopoletin were analyzed in cultured tobacco (Nicotiana tabacum) cells treated with three proteinaceous elicitors: two elicitins (alpha-megaspermin and beta-megaspermin) and one glycoprotein. These three proteins have been isolated from Phytophthora megasperma H20 and have been previously shown to be equally efficient in inducing a hypersensitive response (HR) upon infiltration into tobacco leaves. However, in cultured tobacco cells these elicitors exhibited strikingly different biological activities. beta-Megaspermin was the only elicitor that caused cell death and induced a strong, biphasic H(2)O(2) burst. Both elicitins stimulated PAL activity similarly and strongly, while the glycoprotein caused only a slight increase. Only elicitins induced SA accumulation and scopoletin consumption, and beta-megaspermin was more efficient. To assess the role of H(2)O(2) in HR cell death and defense response expression in elicitin-treated cells, a gain and loss of function strategy was used. Our results indicated that H(2)O(2) was neither necessary nor sufficient for HR cell death, PAL activation, or SA accumulation, and that extracellular H(2)O(2) was not a direct cause of intracellular scopoletin consumption. (+info)
Phosphorylation of phenylalanine ammonia-lyase: evidence for a novel protein kinase and identification of the phosphorylated residue.
The site of phosphorylation of phenylalanine ammonia-lyase (PAL) has been identified as a threonine residue. A Ca(2+)-stimulated protein kinase of approximately 55 kDa has been partially purified from elicited cells. The kinase can phosphorylate a synthetic peptide derived from PAL and a recombinant poplar PAL. PAL phosphorylation was associated with a decrease in Vmax in agreement with the suggestion that protein phosphorylation is involved in marking PAL subunits for turnover. The phosphorylation site in French bean PAL is most likely Thr545 in the sequence VAKRTLTT (539-546). Conservation of the phosphorylation site in PAL from diverse species suggests that phosphorylation of PAL may be a ubiquitous regulatory mechanism in higher plants. (+info)
Overexpression of a designed 2.2 kb gene of eukaryotic phenylalanine ammonia-lyase in Escherichia coli.
Phenylalanine ammonia-lyase (EC 188.8.131.52) is a key enzyme in the secondary metabolism of higher plants catalyzing the non-oxidative conversion of L-phenylalanine into transcinnamate. The nucleotide sequence of its 2.2 kb gene was designed for expression in Escherichia coli and synthesized in a single reaction from 108 oligonucleotides using assembly PCR. After amplification, the gene was cloned into the expression vector pT7-7 and coexpressed with the chaperone HSP-60 system. The expression system yielded 70 mg of fully active enzyme per liter culture. (+info)
Altering expression of cinnamic acid 4-hydroxylase in transgenic plants provides evidence for a feedback loop at the entry point into the phenylpropanoid pathway.
Pharmacological evidence implicates trans-cinnamic acid as a feedback modulator of the expression and enzymatic activity of the first enzyme in the phenylpropanoid pathway, L-phenylalanine ammonia-lyase (PAL). To test this hypothesis independently of methods that utilize potentially non-specific inhibitors, we generated transgenic tobacco lines with altered activity levels of the second enzyme of the pathway, cinnamic acid 4-hydroxylase (C4H), by sense or antisense expression of an alfalfa C4H cDNA. PAL activity and levels of phenylpropanoid compounds were reduced in leaves and stems of plants in which C4H activity had been genetically down-regulated. However, C4H activity was not reduced in plants in which PAL activity had been down-regulated by gene silencing. In crosses between a tobacco line over-expressing PAL from a bean PAL transgene and a C4H antisense line, progeny populations harboring both the bean PAL sense and C4H antisense transgenes had significantly lower extractable PAL activity than progeny populations harboring the PAL transgene alone. Our data provide genetic evidence for a feedback loop at the entry point into the phenylpropanoid pathway that had previously been inferred from potentially artifactual pharmacological experiments. (+info)
UV light selectively coinduces supply pathways from primary metabolism and flavonoid secondary product formation in parsley.
The UV light-induced synthesis of UV-protective flavonoids diverts substantial amounts of substrates from primary metabolism into secondary product formation and thus causes major perturbations of the cellular homeostasis. Results from this study show that the mRNAs encoding representative enzymes from various supply pathways are coinduced in UV-irradiated parsley cells (Petroselinum crispum) with two mRNAs of flavonoid glycoside biosynthesis, encoding phenylalanine ammonia-lyase and chalcone synthase. Strong induction was observed for mRNAs encoding glucose 6-phosphate dehydrogenase (carbohydrate metabolism, providing substrates for the shikimate pathway), 3-deoxyarabinoheptulosonate 7-phosphate synthase (shikimate pathway, yielding phenylalanine), and acyl-CoA oxidase (fatty acid degradation, yielding acetyl-CoA), and moderate induction for an mRNA encoding S-adenosyl-homocysteine hydrolase (activated methyl cycle, yielding S-adenosyl-methionine for B-ring methylation). Ten arbitrarily selected mRNAs representing various unrelated metabolic activities remained unaffected. Comparative analysis of acyl-CoA oxidase and chalcone synthase with respect to mRNA expression modes and gene promoter structure and function revealed close similarities. These results indicate a fine-tuned regulatory network integrating those functionally related pathways of primary and secondary metabolism that are specifically required for protective adaptation to UV irradiation. Although the response of parsley cells to UV light is considerably broader than previously assumed, it contrasts greatly with the extensive metabolic reprogramming observed previously in elicitor-treated or fungus-infected cells. (+info)