Extraction of phenylalanine ammonia lyase from Anabaena variabilis isolates from Iran

Context: Phenylketonuria (PKU) is the most common hereditary defect of phenylalanine hydroxylase (PAH) enzyme achieving the hydroxylation of phenylalanine (Phe). Phenylalanine ammonia lyase (PAL) converts Phe to a harmless metabolite, trans-cinnamic acid (TCA) in plants and PAL enzyme activity is fairly high in plants rich in flavonoids.Objective: The study aimed the biochemical analysis of PAL form Centaurea depressa BIEB. (Asteraceae) a flavonoid rich plant. This study may form the main frame of future research efforts for the development of a plant preparation aimed for oral intake in PKU patients in an attempt to enrich their diet by allowing them to ingest some food stuff containing Phe without being exposed to complications.Materials and methods: PAL was partially purified from the leaves of C. depressa. Enzyme activity was determined in comparison with that of other herbs that reportedly have a high PAL activity. Enzyme optimization was achieved and the PAL protein was detected by western blotting.Results: C. depressa PAL demonstrated high activity (34.9 ± 0.6 U/mg protein). The enzyme was purified by 1.92-fold, which resulted in an activity of 53.30 ± 0.2 U/mg protein. The high-performance liquid chromatography analyzes of the PAL activity both before and after purification were in agreement. Western blot of PAL exhibited a 70 kDa protein band. The optimum pH and temperature are pH 8.8 and 37 °C. The optimum activities under gastric and intestinal digestion conditions were observed at pH 4.0 and pH 8.0, respectively.Discussion and conclusion: PAL activity of C. depressa is high, and does not disappear under different environmental conditions. This enzyme could be used for the development of dietary foods and biotechnological products for patients with PKU.

Phenylketonuria (PKU) is an inherited disorder that increases the levels of an amino acid phenylalanine in the blood. Phenylalanine is found in all proteins and in some artificial sweeteners. Phenylketonuria is caused by mutations in the PAH gene, responsible for synthesis of the enzyme phenylalanine hydroxylase. This enzyme converts the amino acid phenylalanine (PA) into tyrosine, which is also very important amino acid for the human body. The gene mutations reduce the activity of the enzyme. As a result the levels of PA in the blood increase and can cause a brain damage. There is no cure for PKU, but treatment can prevent intellectual disabilities and other health problems. People with PKU need to follow a diet that limits food with PA. The diet should be followed carefully and be started as soon after the birth as possible. Experts recommend that people with PKU stay on the diet throughout their lives for better physical and mental health. The U.S. Food and Drug Administration (FDA) has approved the drug sapropterin dihydrochloride (Kuvan) for the treatment of PKU. It is a form of BH4, which is a substance in the body that helps to break down phe-nylalanine. But even if the medication helps, it will not decrease the PA to the desired amount and must be used together with the PKU diet. If people with PKU do not restrict the phenylalanine in their diet, they develop severe intellectual and physical disabilities.

Phenylketonuria (PKU) is a metabolic disorder, in which loss of phenylalanine hydroxylase activity results in neurotoxic levels of phenylalanine. We used the Pah(enu2/enu2) PKU mouse model in short- and long-term studies of enzyme substitution therapy with PEGylated phenylalanine ammonia lyase (PEG-PAL conjugates) from 4 different species. The most therapeutically effective PAL (Av, Anabaena variabilis) species was one without the highest specific activity, but with the highest stability; indicating the importance of protein stability in the development of effective protein therapeutics. A PEG-Av-p.C503S/p.C565S-PAL effectively lowered phenylalanine levels in both vascular space and brain tissue over a >90 day trial period, resulting in reduced manifestations associated with PKU, including reversal of PKU-associated hypopigmentation and enhanced animal health. Phenylalanine reduction occurred in a dose- and loading-dependent manner, and PEGylation reduced the neutralizing immune response to the enzyme. Human clinical trials with PEG-Av-p.C503S/p.C565S-PAL as a treatment for PKU are underway.

In phenylketonuria (PKU) patients, a genetic defect in the enzyme phenylalanine hydroxylase (PAH) leads to elevated systemic phenylalanine (Phe), which can result in severe neurological impairment. As a treatment for PKU, Escherichia coli Nissle (EcN) strain SYNB1618 was developed under Synlogic’s Synthetic Biotic™ platform to degrade Phe from within the gastrointestinal (GI) tract. This clinical-stage engineered strain expresses the Phe-metabolizing enzyme phenylalanine ammonia lyase (PAL), catalyzing the deamination of Phe to the non-toxic product trans-cinnamate (TCA). In the present work, we generate a more potent EcN-based PKU strain through optimization of whole cell PAL activity, using biosensor-based high-throughput screening of mutant PAL libraries. A lead enzyme candidate from this screen is used in the construction of SYNB1934, a chromosomally integrated strain containing the additional Phe-metabolizing and biosafety features found in SYNB1618. Head-to-head, SYNB1934 demonstrates an approximate two-fold increase in in vivo PAL activity compared to SYNB1618.

l‐Phenylalanine ammonia‐lyase (EC 4.3.1.9, which catalyses the first reaction in the biosynthesis of plant phenylpropanoid products, undergoes a transient increase in activity in excised sections of pea (Pisum sativum) epicotyl tissue. Exogenous supplies of pathway intermediates inhibit the initial development of phenylalanine ammonia‐lyase activity and, if added at the time of high enzyme levels, cause a rapid decay in enzyme activity. The inhibitory effect of exogenous cinnamic acid, the immediate product of the enzyme, is expressed very rapidly (lag < 1 h) with the initial rate of decay of enzyme activity and the final steady‐state level of enzyme activity being dependent on the concentration of cinnamic acid.The response has been studied by density labelling in vivo with 2H from 2H2O with analysis of the equilibrium distribution of enzyme activity in high‐resolution KBr density gradients. Cinnamic acid, at a concentration of 1 mM completely inhibits further incorporation of label and also markedly stimulates the removal of pre‐existing, unlabelled enzyme. Inhibition of de novo production and stimulation of removal occur at concentrations of cinnarnic acid as low as 10 μM.The initial increase in enzyme activity reflects an increase in the rate of de novo enzyme production against a low background rate of enz:yme removal. The subsequent decay in enzyme activity is a result of a decline in production of the enzyme concomitant with a marked increase in the rate of removal of enzyme activity. In the presence of α‐ aminooxy‐β‐phenylpropionic acid, which specifically inhibits phenylalanine ammonia‐lyase and hence endogenous generation of cinnamic cid, high rates of enzyme production and low rates of enzyme removal are maintained leading to high levels of zyme activity in the later stages of enzyme induction. These data indicate the operation of dual feedback modulation of phenylalanine ammonia‐lyase in vivo following endogenous production of cinnamic acid. Such dual control by a product over both production and removal of an enzyme may be of general importance in slowly growing plant cells for the rapid removal of biosynthetic enzymes no longer required by the cell.

Phenylketonuria (PKU) is a genetic metabolic disease in which the decrease or loss of phenylalanine hydroxylase (PAH) activity results in elevated, neurotoxic levels of phenylalanine (Phe). Due to many obstacles, PAH enzyme replacement therapy is not currently an option. Treatment of PKU with an alternative enzyme, phenylalanine ammonia lyase (PAL), was first proposed in the 1970s. However, issues regarding immunogenicity, enzyme production and mode of delivery needed to be overcome. Through the evaluation of PAL enzymes from multiple species, three potential PAL enzymes from yeast and cyanobacteria were chosen for evaluation of their therapeutic potential. The addition of polyethylene glycol (PEG, MW = 20,000), at a particular ratio to modify the protein surface, attenuated immunogenicity in an animal model of PKU. All three PEGylated PAL candidates showed efficacy in a mouse model of PKU (BTBR Pahenu2) upon subcutaneous injection. However, only PEGylated Anabaena variabilis (Av) PAL-treated mice demonstrated sustained low Phe levels with weekly injection and was the only PAL evaluated that maintained full enzymatic activity upon PEGylation. A PEGylated recombinant double mutant version of AvPAL (Cys503Ser/Cys565Ser), rAvPAL-PEG, was selected for drug development based on its positive pharmacodynamic profile and favorable expression titers. PEGylation was shown to be critical for rAvPAL-PEG efficacy as under PEGylated rAvPAL had a lower pharmacodynamic effect. rAvPAL and rAvPAL-PEG had poor stability at 4°C. L-Phe and trans-cinnamate were identified as activity stabilizing excipients. rAvPAL-PEG is currently in Phase 3 clinical trials to assess efficacy in PKU patients.

<p><em>Glycyrrhiza glabra</em> is an ancient herbal medicine rich in large number of secondary metabolites which attribute to its therapeutic properties. These metabolites are usually obtained from field grown plants and their yields vary greatly on the basis of environmental conditions. Plant tissue culture can thus be a preferred method for consistent production of such metabolites. This study dealt with the enhancement of flavonoids through precursor feeding in callus cultures of <em>G. glabra </em>and investigated the influence of cinnamic acid on phenylalanine ammonia lyase (PAL) activity and production of licochalcone A, liquirtigenin and licoisoflavone B. Unorganized callus cultures were established from young leaf explants on Murashige and Skoog’s (MS) medium supplemented with NAA (1mg/l), BAP (0.5 mg/l) and various concentrations of cinnamic acid. Flavonoids were obtained from calli through solvent extraction and were identified and quantified through Gas-Chromatography Mass spectrometry. Cinnamic acid supplementation at appropriate concentrations (50mg/100ml for licochalcone A and liquirtigenin, and 125mg/100ml for licoisoflavone B) significantly increased their production to 1.28, 1.2 and 9.76 folds respectively. However, prolonged treatment of cinnamic acid at concentration beyond 50mg/100ml led to decrease in the production of liquirtigenin and licochalcone A , but caused fair increase in licoisoflavone B. Also cinnamic acid concentrations higher than 50mg/100ml reduced the activity of PAL enzyme due to its feedback inhibition, but at the same time might have modulated other intermediate enzymes of the pathway like chalcone isomerase favoring the formation of licoisoflavone B. Therefore, this study provides clear evidences of enzymatic regulation of phenylpropanoid pathway by cinnamic acid in <em>G. glabra</em> callus cultures.</p>

Standard therapy for phenylketonuria (PKU), the most common inherited disorder in amino acid metabolism, is an onerous phenylalanine-restricted diet. Adherence to this stringent diet regimen decreases as patients get older, and this lack of adherence is directly associated with cognitive and executive dysfunction and psychiatric issues. These factors emphasize the need for alternative pharmacological therapies to help treat patients with PKU. Sapropterin dihydrochloride is a synthetic form of tetrahydrobiopterin, the cofactor of phenylalanine hydroxylase that in pharmacological doses can stabilize and increase residual enzyme activity in some patients with PKU. About one-third of all patients with PKU respond to oral sapropterin. Phenylalanine ammonia lyase (PAL) is a prokaryotic enzyme that converts phenylalanine to ammonia and trans-cinnamic acid. Phase I and II trials have shown that injectable recombinant Anabaena variabilis PAL produced in Escherichia coli conjugated with PEG can reduce phenylalanine levels in subjects with PKU. The most frequently reported adverse events were injection-site reactions, dizziness and immune reactions. Additionally, oral administration of PAL and delivery of enzyme substitution therapies by encapsulation in erythrocytes are being investigated. Novel therapies for patients with PKU appear to be options to reduce phenylalanine levels, and may reduce the deleterious effects of this disorder.

Pharmacodynamics, safety, tolerability and pharmacokinetics of a single oral dose of an engineered phenylalanine ammonia-lyase in patients with phenylketonuria

Phenylalanine ammonia-lyase (PAL) is the first entry enzyme of the phenylpropanoid pathway that converts phenylalanine to cinnamic acid which is the precursor of various secondary metabolites. PAL is recently formulated for Phenylketonuric patients in pegylated forms. Screening a PAL with the highest affinity to the substrate is of great importance for this purpose. PAL exists in all higher plants and some fungi and few bacteria. Ancestors of land plants have been adopted by evolving metabolic pathways. A multi-gene family encodes PAL by gene duplication events in most plants. In this study, the taxonomic distribution and phylogeny of pal gene found in land plants, fungi and bacteria have been analyzed. It seems that the ancestor of plants acquired a pal gene via horizontal gene transfer in symbioses with bacteria and fungi. Gymnosperms have kept a diverse set of pal genes that arose from gene duplication events. In angiosperms, after the divergence of dicotyledons from monocots, pal genes were duplicated many times. The close paralogues of pal genes in some species indicate expansion of gene families after the divergence in plant pal gene evolution. Interestingly, some of the plant pals clustered by species a way that pals within one species are more closely related to each other than to homologs in the other species which indicates this duplication event occurred more recently.

Phenylalanine ammonia lyase (PAL): From discovery to enzyme substitution therapy for phenylketonuria

Phenylketonuria (PKU) is a rare metabolic disorder characterized by impaired conversion of phenylalanine (Phe) to tyrosine. If left untreated, the resultant accumulation of excess blood Phe can cause physiological, neurological, and intellectual disabilities. The National PKU Alliance (NPKUA) conducted a survey of its membership to assess current health status and interest in new treatments for PKU. Of the 625 survey respondents, less than half (46.7%) reported blood Phe within (120–360μmol/L) — the range recommended by the American College of Medical Genetics and Genomics (ACMG). The survey results also showed that younger (≤18years) individuals were about 3-times as successful in keeping their blood Phe concentrations within the recommended clinical range compared with adults. Blood Phe over 360μmol/L was reported in one-quarter (25.5%) of ≤18year old individuals and almost two-thirds (61.5%) of adults. A little more than half (51.7%) of respondents reported having difficulty in managing their PKU, including the maintenance of a Phe-restricted diet. Individuals with PKU desire new treatments that would allow them to increase their intake of natural protein, discontinue or reduce their intake of medical foods (medical formula and foods modified to be low in protein), improve their mental health (including a reduction in depression and anxiety), and a reduction of their blood Phe concentrations. Respondents preferred oral administration of any newly developed therapies and, in general, disliked therapeutic injections. Injections at home were preferred over injections at a clinic. Payers, government agencies, clinicians, and industry partners should consider patient input when developing and approving new therapies and treatments for PKU.

Treatment of phenylketonuria (PKU) has evolved since the initial introduction of a phenylalanine (Phe) restricted diet. The most recent option for adults affected with PKU is treatment with an alternate enzyme, phenylalanine ammonia lyase (PAL), that metabolizes excess Phe. Proper management of all patients with PKU relies on accurate measurement of Phe levels in blood, to comply with guidance intended to minimize the neurological symptoms. Recently, our laboratory was notified of discrepant results for a patient with PKU who is treated with pegvaliase. Two specimens were collected at the same time but yielded unexpectedly different Phe concentrations. After exclusion of specimen mix-ups or analytical errors, we suspected that there was residual pegvaliase activity in the specimens continuing to degrade Phe after collection. To investigate this possibility, we performed spiking studies that showed the degradation of Phe over time at ambient temperatures. Sample preparation by protein crash appears to deactivate pegvaliase and prevents further Phe degradation. However, because pegvaliase deactivation would be required immediately following blood collection, appropriate mitigation measures must be implemented, including stringent pre-analytical requirements, alternate sample matrices such as dried blood spots, or point of care testing. Until then, health care professionals need to be cautious in their interpretation of Phe levels in their patients with PKU that are treated with pegvaliase.

eP276: Development of an overdue outreach program to improve adherence to phenylketonuria management

Biomedical applications of microbial phenylalanine ammonia lyase: Current status and future prospects