Comparative analyses reveal a phenylalanine ammonia lyase dependent and salicylic acid mediated host resistance in Zingiber zerumbet against the necrotrophic soft rot pathogen Pythium myriotylum.

Little is known about the molecular basis of host defense in resistant wild species Zingiber zerumbet (L.) Smith against the soil-borne, necrotrophic oomycete pathogen Pythium myriotylum Drechsler, which causes the devastating soft rot disease in the spice crop ginger (Zingiber officinale Roscoe). We investigated the pattern of host defense between Z. zerumbet and ginger in response to P. myriotylum inoculation. Analysis of gene expression microarray data revealed enrichment of phenylpropanoid biosynthetic genes, particularly lignin biosynthesis genes, in pathogen-inoculated Z. zerumbet compared to ginger. RT-qPCR analysis showed the robust activation of phenylpropanoid biosynthesis genes in Z. zerumbet, including the core genes PAL, C4H, 4CL, and the monolignol biosynthesis and polymerization genes such as CCR, CAD, C3H, CCoAOMT, F5H, COMT, and LAC. Additionally, Z. zerumbet exhibited the accumulation of the phenolic acids including p-coumaric acid, sinapic acid, and ferulic acid that are characteristic of the cell walls of commelinoid monocots like Zingiberaceae and are involved in cell wall strengthening by cross linking with lignin. Z. zerumbet also had higher total lignin and total phenolics content compared to pathogen-inoculated ginger. Phloroglucinol staining revealed the enhanced fortification of cell walls in Z. zerumbet, specifically in xylem vessels and surrounding cells. The trypan blue staining indicated inhibition of pathogen growth in Z. zerumbet at the first leaf whorl, while ginger showed complete colonization of the pith within 36 h post inoculation (hpi). Accumulation of salicylic acid (SA) and induction of SA regulator NPR1 and the signaling marker PR1 were observed in Z. zerumbet. Silencing of PAL in Z. zerumbet through VIGS suppressed downstream genes, leading to reduced phenylpropanoid accumulation and SA level, resulting in the susceptibility of plants to P. myriotylum. These findings highlight the essential role of PAL-dependent mechanisms in resistance against P. myriotylum in Z. zerumbet. Moreover, our results suggest an unconventional role for SA in mediating host resistance against a necrotroph. Targeting the phenylpropanoid pathway could be a promising strategy for the effective management of P. myriotylum in ginger.

Study on the biocontrol effect and physiological mechanism of Hannaella sinensis on the blue mold decay of apples.

Blue mold decay is a major postharvest disease of apples, causing considerable losses to the apple industry. In the early stage of this research, an antagonistic yeast, Hannaella sinensis, with a good control effect on the blue mold of apples, was selected. On this basis, the main purpose of this work was to study the biocontrol effect of H. sinensis on the blue mold of apples and the mechanisms involved. The results showed that H. sinensis could effectively control the blue mold decay of apples, reduce the rot rate and diameter, and the antagonistic effect strengthened with the increase of H. sinensis concentration (1 × 108 cells/mL). Further in vitro experiments proved that H. sinensis could significantly inhibit the spore germination and germ tube length of P. expansum. In addition, stable colonization of H. sinensis on apple wounds and surfaces confirmed the environmental adaptability and the ability to compete with other microbiota for nutrition and space. Moreover, H. sinensis induced the activities of resistance-related enzymes such as polyphenol oxidase (PPO), peroxidase (POD), ascorbate peroxidase (APX), superoxide dismutase (SOD), and phenylalanine ammonia-lyase (PAL) in apples and the content of the coding genes corresponding to these enzymes was also higher than that of the control group. Our results indicate that H. sinensis treatment could induce the disease resistance of apples. In summary, H. sinensis served as a promising antagonistic yeast for the prevention and treatment of postharvest blue mold decay of apples.

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

Phenylalanine ammonia lyase (PAL) has recently emerged as an important therapeutic enzyme with several biomedical applications. The enzyme catabolizes l-phenylalanine to trans-cinnamate and ammonia. PAL is widely distributed in higher plants, some algae, ferns, and microorganisms, but absent in animals. Although microbial PAL has been extensively exploited in the past for producing industrially important metabolites, its high substrate specificity and catalytic efficacy lately spurred interest in its biomedical applications. PEG-PAL drug named Palynziq™, isolated from Anabaena variabilis has been recently approved for the treatment of adult phenylketonuria (PKU) patients. Further, it has exhibited high potency in regressing tumors and treating tyrosine related metabolic abnormalities like tyrosinemia. Several therapeutically valuable metabolites have been biosynthesized via its catalytic action including dietary supplements, antimicrobial peptides, aspartame, amino-acids, and their derivatives. This review focuses on all the prospective biomedical applications of PAL. It also provides an overview of the structure, production parameters, and various strategies to improve the therapeutic potential of this enzyme. Engineered PAL with improved pharmacodynamic and pharmacokinetic properties will further establish this enzyme as a highly efficient biological drug.

An R2R3 MYB transcription factor confers brown planthopper resistance by regulating the phenylalanine ammonia-lyase pathway in rice.

Brown planthopper (BPH) is one of the most destructive insects affecting rice (Oryza sativa L.) production. Phenylalanine ammonia-lyase (PAL) is a key enzyme involved in plant defense against pathogens, but the role of PAL in insect resistance is still poorly understood. Here we show that expression of the majority of PALs in rice is significantly induced by BPH feeding. Knockdown of OsPALs significantly reduces BPH resistance, whereas overexpression of OsPAL8 in a susceptible rice cultivar significantly enhances its BPH resistance. We found that OsPALs mediate resistance to BPH by regulating the biosynthesis and accumulation of salicylic acid and lignin. Furthermore, we show that expression of OsPAL6 and OsPAL8 in response to BPH attack is directly up-regulated by OsMYB30, an R2R3 MYB transcription factor. Taken together, our results demonstrate that the phenylpropanoid pathway plays an important role in BPH resistance response, and provide valuable targets for genetic improvement of BPH resistance in rice.

Pegvaliase: a novel treatment option for adults with phenylketonuria.

OBJECTIVE: In May 2018, the US Food and Drug Administration approved pegvaliase-pqpz (Palynziq*), the first enzyme substitution therapy for the treatment of phenylketonuria (PKU). This article provides an overview of the mechanism of action, pharmacokinetic properties, clinical efficacy, and the safety and tolerability profile of pegvaliase. METHODS: Relevant information was identified through a comprehensive literature search of several databases using the keywords pegvaliase, rAvPAL-PEG, and phenylketonuria. Additional information was gathered from the pegvaliase package insert, posters presented at scientific meetings, and materials provided from the manufacturer, BioMarin. RESULTS: Pegvaliase is effective in decreasing blood phenylalanine levels, and is associated with a manageable side-effect profile. Phase III clinical trial data demonstrated that 60.7% of patients were able to achieve blood phenylalanine levels less than the guideline recommended 360 µmol/L at 24 months. Brief sub-studies also showed the improvement in inattention symptoms in patients treated with pegvaliase, compared to placebo. CONCLUSION: Pegvaliase is a promising new treatment option for adults living with PKU. Further studies are warranted to determine long-term safety and clinical efficacy in sub-populations.

Descriptive and hedonic analyses of low-Phe food formulations containing corn (Zea mays) seedling roots: toward development of a dietary supplement for individuals with phenylketonuria.

BACKGROUND: Seedling roots of anthocyanin-rich corn (Zea mays) cultivars contain high levels of phenylalanine ammonia lyase (PAL) activity. The development of a natural dietary supplement containing corn roots could provide the means to improve the restrictive diet of phenylketonuria (PKU) patients by increasing their tolerance to dietary phenylalanine (Phe). Therefore this research was undertaken to explore the sensory characteristics of roots of four corn cultivars as well as to develop and evaluate food products (cereal bar, beverage, jam-like spread) to which roots had been added. RESULTS: Sensory profiles of corn roots were investigated using ten trained judges. Roots of Japanese Striped corn seedlings were more bitter, pungent and astringent than those of white and yellow cultivars, while roots from the Blue Jade cultivar had a more pronounced earthy/mushroom aroma. Consumer research using 24 untrained panelists provided hedonic (degree-of-liking) assessments for products with and without roots (controls). The former had lower mean scores than the controls; however, the cereal bar had scores above 5 on the nine-point scale for all hedonic assessments compared with the other treated products. CONCLUSION: By evaluating low-Phe food products containing corn roots, this research ascertained that the root-containing low-Phe cereal bar was an acceptable 'natural' dietary supplement for PKU-affected individuals.

Single-dose, subcutaneous recombinant phenylalanine ammonia lyase conjugated with polyethylene glycol in adult patients with phenylketonuria: an open-label, multicentre, phase 1 dose-escalation trial.

BACKGROUND: Phenylketonuria is an inherited disease caused by impaired activity of phenylalanine hydroxylase, the enzyme that converts phenylalanine to tyrosine, leading to accumulation of phenylalanine and subsequent neurocognitive dysfunction. Phenylalanine ammonia lyase is a prokaryotic enzyme that converts phenylalanine to ammonia and trans-cinnamic acid. We aimed to assess the safety, tolerability, pharmacokinetic characteristics, and efficacy of recombinant Anabaena variabilis phenylalanine ammonia lyase (produced in Escherichia coli) conjugated with polyethylene glycol (rAvPAL-PEG) in reducing phenylalanine concentrations in adult patients with phenylketonuria. METHODS: In this open-label, phase 1, multicentre trial, single subcutaneous injections of rAvPAL-PEG were given in escalating doses (0·001, 0·003, 0·010, 0·030, and 0·100 mg/kg) to adults with phenylketonuria. Participants aged 18 years or older with blood phenylalanine concentrations of 600 µmol/L or higher were recruited from among patients attending metabolic disease clinics in the USA. The primary endpoints were safety and tolerability of rAvPAL-PEG. Secondary endpoints were the pharmacokinetic characteristics of the drug and its effect on concentrations of phenylalanine. Participants and investigators were not masked to assigned dose group. This study is registered with ClinicalTrials.gov, number NCT00925054. FINDINGS: 25 participants were recruited from seven centres between May 6, 2008, and April 15, 2009, with five participants assigned to each escalating dose group. All participants were included in the safety population. The most frequently reported adverse events were injection-site reactions and dizziness, which were self-limited and without sequelae. Two participants had serious adverse reactions to intramuscular medroxyprogesterone acetate, a drug that contains polyethylene glycol as an excipient. Three of five participants given the highest dose of rAvPAL-PEG (0·100 mg/kg) developed a generalised skin rash. By the end of the study, all participants had developed antibodies against polyethylene glycol, and some against phenylalanine ammonia lyase as well. Drug concentrations peaked about 89-106 h after administration of the highest dose. Treatment seemed to be effective at reducing blood phenylalanine in all five participants who received the highest dose (mean reduction of 54·2% from baseline), with a nadir about 6 days after injection and an inverse correlation between drug and phenylalanine concentrations in plasma. Phenylalanine returned to near-baseline concentrations about 21 days after the injection. INTERPRETATION: Subcutaneous administration of rAvPAL-PEG in a single dose of up to 0·100 mg/kg was fairly safe and well tolerated in adult patients with phenylketonuria. At the highest dose tested, rAvPAL-PEG reduced blood phenylalanine concentrations. In view of the development of antibodies against polyethylene glycol (and in some cases against phenylalanine ammonia lyase), future studies are needed to assess the effect of repeat dosing. FUNDING: BioMarin Pharmaceutical.

Recombinant l-phenylalanine ammonia lyase from Rhodosporidium toruloides as a potential anticancer agent.

The recombinant producer strain expressing Rhodosporidium toruloides l-phenylalanine ammonia lyase (PAL) has been obtained, and a purification procedure of PAL has been developed. The purified enzyme, PAL, has the following biochemical and catalytic characteristics: Km for l-Phe of 0.49 mM, pH optimum at 8.5, and temperature optimum at 50°C. PAL exhibited a significant cytotoxic effect toward the following cell lines: MCF7 (IC50 = 1.97 U/mL), DU145 (IC50 = 7.3 U/mL), which are comparable with E. coli l-asparaginase type-II cytotoxicity in vitro. Administration of PAL (200-400 U/kg) to L5178y-bearing mice for five times (a total dose of 1000-2000 U/kg) was well tolerated and showed the increase of life span (ILS) = 12-16%, P < 0.05. Data obtained suggest that PAL from R. toruloides has a potential for cancer treatment.

Control of postharvest soft rot caused by Erwinia carotovora of vegetables by a strain of Bacillus amyloliquefaciens and its potential modes of action.

Erwinia carotovora subsp. carotovora (Ecc), the causal agent of bacterial soft rot, is one of the destructive pathogens of postharvest vegetables. In this study, a bacterial isolate (BGP20) from the vegetable farm soil showed strong antagonistic activity against Ecc in vitro, and its twofold cell-free culture filtrate showed excellent biocontrol effect in controlling the postharvest bacterial soft rot of potatoes at 25 °C. The anti-Ecc metabolites produced by the isolate BGP20 had a high resistance to high temperature, UV-light and protease K. Based on the colonial morphology, cellular morphology, sporulation, and partial nucleotide sequences of 16S rRNA and gyrB gene, the isolate BGP20 was identified as Bacillus amyloliquefaciens subsp. plantarum. Further in vivo assays showed that the BGP20 cell culture was more effective in controlling the postharvest bacterial soft rot of green peppers and Chinese cabbages than its twofold cell-free culture filtrate. In contrast, the biocontrol effect and safety of the BGP20 cell culture were very poor on potatoes. In the wounds of potatoes treated with both the antagonist BGP20 and the pathogen Ecc, the viable count of Ecc was 31,746 times that of BGP20 at 48 h of incubation at 25 °C. But in the wounds of green peppers, the viable count of BGP20 increased 182.3 times within 48 h, and that of Ecc increased only 51.3 %. In addition, the treatment with both BGP20 and Ecc induced higher activity of phenylalanine ammonia-lyase (PAL) than others in potatoes. But the same treatment did not induce an increase of PAL activity in green peppers. In conclusion, the present study demonstrated that the isolate BGP20 is a promising candidate in biological control of postharvest bacterial soft rot of vegetables, but its main mode of action is different among various vegetables.

Preclinical evaluation of multiple species of PEGylated recombinant phenylalanine ammonia lyase for the treatment of phenylketonuria.

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.

Effect of mycorrhizal fungi on some defense enzymes against Gaeumannomyces gaminis in wheat.

At this research, the effect of mycorrhizal fungi (Glomus etunicatum) On Pero Xidase (POX) and Phenylalanine Ammonia Lyase (PAL) activities and isozymatic pattern against Gaeumannomyces graminis were studied in wheat plants. Seeds were planted in inoculated soils in 4 treatment groups including Control (C), Mycorrhiza (M), Pathogen (P) and Pathogen- Mycorrhiza (PM). Plants were harvested 17 days after inoculation. POX activities in PM group were significantly greater than control group. Significant differences were not observed between P and C groups. POX activities significantly decreased in M group. PAL activities in M group were significantly greater than other groups. PAL activities in P and PM groups were significantly greater than C group. The appearance of new isozyme was induced in PM group. It is highly probable that induced POX isozymic activity and/or appearance of new isozymes may be responsible for elevated POX activity. Present results showed that the isozymatic patterns of POX were changed by inoculation ofmycorrhiza and/or pathogen. The obtained results from this research is agreement with other researches about the enhancing effect of mycorrhizal fungi on PAL activity. The obtained results from the present research, confirm this opinion that defense related proteins is not induced in compatible interactions or is weak.

Evaluation of possible reasons for the low phenylalanine ammonia lyase activity in cellulose nitrate membrane microcapsules.

Microencapsulated phenylalanine ammonia lyase (PAL) exhibits a marked reduction in activity compared to the activity of the free enzyme in pH 8.5 Tris buffer. The purpose of this investigation was to evaluate the contribution of incomplete entrapment, the internal environment of cellulose nitrate membrane microcapsules, the diffusional barrier of the membrane and the microcapsulation process to the low activity of encapsulated PAL. A solution of PAL and 10% w/v hemoglobin was incorporated into cellulose nitrate membrane microcapsules. Hemoglobin incorporation was used as a surrogate marker of PAL entrapment. Using 14C hemoglobin, the encapsulation efficiency was determined to be 70% and suggested that incomplete entrapment might partially account for the low activity of encapsulated PAL. The effect of the internal environment of the microcapsule (10% hemoglobin solution) on PAL activity was evaluated by comparing enzyme activity in 10% w/v hemoglobin solution and pH 8.5 Tris buffer. Similar K(M) and V(max) values of PAL in the two media indicated that the internal environment of the microcapsule did not contribute to the reduction in activity of the encapsulated enzyme. The contribution of a membrane diffusional barrier was determined by breaking the putative barrier and measuring PAL activity in intact and broken microcapsules. Similar activity of PAL in these two conditions is evidence for the lack of a diffusional barrier. The effect of the microencapsulation process on PAL activity was evaluated by comparing K(M) and V(max) of free and encapsulated PAL. Similar K(M) values in these two media suggested that the process did not affect the conformation of PAL. However, encapsulated PAL had a 50% lower V(max) value compared to free PAL, which showed that the microencapsulation process deactivated a substantial proportion of the enzyme.

Post-transcriptional regulation of phenylalanine ammonia-lyase expression in tobacco following recovery from gene silencing.

Introduction of a bean phenylalanine ammonia-lyase (PAL) transgene into tobacco plants results in epigenetic post-transcriptional gene silencing which is unstable, such that after self-pollination first generation progeny may become PAL over-expressors. The change from gene silencing to PAL over-expression is accompanied by a loss of cytosine methylation of the PAL transgene and reduced methylation of the endogenous tobacco PAL2 gene, but not the PAL1 gene. These changes are associated with the appearance of high levels of bean PAL and tobacco PAL2 transcripts in the total RNA fraction from PAL over-expressing plants. However, tobacco PAL2 transcripts are inefficiently recruited into polysomes, and tobacco PAL2 protein is not detected in leaves of PAL over-expressing or wild-type lines. Thus, in spite of the post-transcriptionally controlled increase in tobacco PAL2 transcripts in PAL over-expressors, the increased PAL activity is primarily the result of the increase in bean PAL transcripts and corresponding enzymatic activity. These results reveal a complex cross-talk between expression of the PAL transgene and the corresponding endogenous PAL genes at the levels of transcription, transcript stability and polysomal recruitment during sense transgene-mediated silencing and subsequent over-expresson of PAL in tobacco.

Phenylalanine ammonia lyase in the management of phenylketonuria: the relationship between ingested cinnamate and urinary hippurate in humans.

The hypothesis that the action of an oral preparation of the enzyme phenylalanine ammonia lyase, used to lower blood phenylalanine levels in phenylketonuria, is by removal of phenylalanine from hydrolysed protein in the lumen of the gut has been examined. Phenylalanine ammonia lyase mediates the conversion of phenylalanine to cinnamate. If cinnamate is produced in the gut it will be absorbed and converted to hippurate in the liver. However, treatment with phenylalanine ammonia lyase does not result in raised urinary hippurate ammonia lyase does not result in raised urinary hippurate levels while ingestion of cinnamate does.

Biochemical properties and immunogenicity of L-phenylalanine ammonia-lyase: effects on tumor-bearing mice.

L-Phenylalanine ammonia-lyase (PAL) from yeast was used to deplete plasma L-phenylalanine and L-tyrosine in an attempt to achieve inhibition of tumor growth in mice. Plasma L-phenylalanine and L-tyrosine were reduced to nondetectable levels when circulating PAL activity was maintained at greater than or equal to 0.06 unit/ml. Repeated administration resulted in the appearance of anti-PAL antibodies. A radioimmunoassay based on the method of Farr was developed to determine quantitatively the presence of anti-PAL. Sublethal total-body irradiation temporarily suppressed the immunologic response of the host. Long-term specific immunosuppression to PAL was achieved with cyclophosphamide (CPA). A single dose of 180 mg/kg of CPA administered ip to mice 24 hours before, simultaneously with, or 24 hours after 100 units/kg of PAL induced tolerance for 450 days (20 injections of enzyme). The plasma half-life of PAL in CPA-treated mice remained essentially the same as that found after a single injection (25 hours), and anti-PAL probably will require specific immunosuppression of the host to repeated injections of the enzyme.

Effects of rapid depletion of phenylalanine and tyrosine on sleep and behavior.

The effects of fluctuations of free amino acid concentrations in plasma on sleep patterns and operant behavior in the squirrel monkey were studied. Plasma phenylalanine (PHE) and tyrosine (TYR) were rapidly lowered to trace levels within 4 hr by intraperitoneal administration of phenylalanine ammonia-lyase (PAL), an enzyme which specifically deaminates both PHE and TYR to inactivate products. Significant alterations in sleep patterns and in performance on a chained operant task involving hold and reaction time components were found, but no significant effect on the performance of a simple operant task was observed. Adminstration of saline or trans-p-cinnamic acid and trans-p-coumaric acid, the products of PHE and TYR deamination, produced no changes in behavior or sleep patterns. The reduction of plasma PHE and TYR resulted in a significant decrease in PHE and TYR levels in whole rat brain. Brain serotonin levels were increased within 4 hr after PAL administration, whereas, dopamine and norepinephrine levels were decreased subsequently (within 8 hr). These studies suggest that circulating levels of PHE and TYR are involved directly or indirectly in the modulation of certain parameters of brain function.