Directed evolution of Anabaena variabilis phenylalanine ammonia-lyase (PAL) identifies mutants with enhanced activities.
ABSTRACT: There is broad interest in engineering phenylalanine ammonia-lyase (PAL) for its biocatalytic applications in industry and medicine. While site-specific mutagenesis has been employed to improve PAL stability or substrate specificity, combinatorial techniques are poorly explored. Here, we report development of a directed evolution technique to engineer PAL enzymes. Central to this approach is a high-throughput enrichment that couples E. coli growth to PAL activity. Starting with the PAL used in the formulation of pegvaliase for PKU therapy, we report previously unidentified mutations that increase turnover frequency almost twofold after only a single round of engineering.
Project description:We have recently observed promising success in a mouse model for treating the metabolic disorder phenylketonuria with phenylalanine ammonia lyase (PAL) from Rhodosporidium toruloides and Anabaena variabilis. Both molecules, however, required further optimization in order to overcome problems with protease susceptibility, thermal stability, and aggregation. Previously, we optimized PAL from R. toruloides, and in this case we reduced aggregation of the A. variabilis PAL by mutating two surface cysteine residues (C503 and C565) to serines. Additionally, we report the structural and biochemical characterization of the A. variabilis PAL C503S/C565S double mutant and carefully compare this molecule with the R. toruloides engineered PAL molecule. Unlike previously published PAL structures, significant electron density is observed for the two active-site loops in the A. variabilis C503S/C565S double mutant, yielding a complete view of the active site. Docking studies and N-hydroxysuccinimide-biotin binding studies support a proposed mechanism in which the amino group of the phenylalanine substrate is attacked directly by the 4-methylidene-imidazole-5-one prosthetic group. We propose a helix-to-loop conformational switch in the helices flanking the inner active-site loop that regulates accessibility of the active site. Differences in loop stability among PAL homologs may explain the observed variation in enzyme efficiency, despite the highly conserved structure of the active site. A. variabilis C503S/C565S PAL is shown to be both more thermally stable and more resistant to proteolytic cleavage than R. toruloides PAL. Additional increases in thermal stability and protease resistance upon ligand binding may be due to enhanced interactions among the residues of the active site, possibly locking the active-site structure in place and stabilizing the tetramer. Examination of the A. variabilis C503S/C565S PAL structure, combined with analysis of its physical properties, provides a structural basis for further engineering of residues that could result in a better therapeutic molecule.
Project description:BACKGROUND:Deficiency of phenylalanine hydroxylase causes phenylketonuria (PKU) with elevated phenylalanine (Phe) levels and associated neuropsychiatric and neurocognitive symptoms. Pegvaliase (PEGylated phenylalanine ammonia lyase) is an investigational agent to lower plasma Phe in adults with PKU. This study aimed to characterize the long-term efficacy, safety, and immunogenicity of pegvaliase in adults with PKU. METHODS:PAL-003 is an ongoing, open-label, long-term extension study of the pegvaliase dose-finding parent phase 2 studies. Participants continued the dose of pegvaliase from one of three parent studies, with dose adjustments to achieve a plasma Phe concentration between 60 and 600 ?mol/L. RESULTS:Mean (standard deviation [SD]) plasma Phe at treatment-naïve baseline for 80 participants in the parent studies was 1302.4 (351.5) ?mol/L. In the 68 participants who entered the extension study, plasma Phe decreased 58.9 (39)% from baseline, to 541.6 (515.5) ?mol/L at Week 48 of treatment. Plasma Phe concentrations ?120 ?mol/L, ?360 ?mol/L, and???600 ?mol/L were achieved by 78.7, 80.0, and 82.5% of participants, respectively. Mean (SD) protein intake at baseline was 69.4 (40.4) g/day (similar to the recommended intake for the unaffected population) and remained stable throughout the study. All participants experienced adverse events (AEs), which were limited to mild or moderate severity in most (88.8%); the most common AEs were injection-site reaction (72.5%), injection-site erythema (67.5%), headache (67.5%), and arthralgia (65.0%). The AE rate decreased from 58.3 events per person-year in the parent studies to 18.6 events per person-year in the extension study. CONCLUSIONS:Pegvaliase treatment in adults with PKU produced meaningful and persistent reductions in mean plasma Phe concentration with a manageable safety profile for most subjects that continued with long-term treatment. TRIAL REGISTRATION:ClinicalTrials.gov , NCT00924703. Registered June 18, 2009, https://clinicaltrials.gov/ct2/show/NCT00924703.
Project description: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 126.96.36.199) to degrade phenylalanine, the harmful nutrient in PKU; in this treatment, PAL acts as a substitute for the enzyme phenylalanine monooxygenase (EC 188.8.131.52), 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.
Project description:The effect of extended reaction times on the regio- and enantioselectivity of the phenylalanine ammonia lyase (PAL)-catalysed amination of a subset of cinnamate derivatives was investigated. This was done using a PAL from the cyanobacterium Anabaena variabilis and incubation in a concentrated ammonia buffer. Whilst early time point analyses revealed excellent selectivities to give mostly the well-documented (S)-α-amino acid products, subsequent accumulation of other regio-/stereo- isomers was seen. For many para-substituted substrates, the β-regioisomer, a previously-unreported product with this enzyme class, was found to become more abundant than the α-, after sufficient incubation, with slight preference for the (R)-enantiomer. Although attempts to tune the selectivity of the PAL toward any of the three side products were largely unsuccessful, the results provide insight into the evolutionary history of this class of enzymes and reinforce the prominence of the toolbox of specific and selective cinnamate-aminating enzymes.
Project description:BACKGROUND:This study assessed the immunogenicity of pegvaliase (recombinant Anabaena variabilis phenylalanine [Phe] ammonia lyase [PAL] conjugated with polyethylene glycol [PEG]) treatment in adults with phenylketonuria (PKU) and its impact on safety and efficacy. METHODS:Immunogenicity was assessed during induction, upward titration, and maintenance dosing regimens in adults with PKU (n?=?261). Total antidrug antibodies (ADA), neutralizing antibodies, immunoglobulin (Ig) M and IgG antibodies against PAL and PEG, IgG and IgM circulating immune complex (CIC) levels, complement components 3 and 4 (C3/C4), plasma Phe, and safety were assessed at baseline and throughout the study. Pegvaliase-specific IgE levels were measured in patients after hypersensitivity adverse events (HAE). FINDINGS:All patients developed ADA against PAL, peaking by 6?months and then stabilizing. Most developed transient antibody responses against PEG, peaking by 3?months, then returning to baseline by 9?months. Binding of ADA to pegvaliase led to CIC formation and complement activation, which were highest during early treatment. Blood Phe decreased over time as CIC levels and complement activation declined and pegvaliase dosage increased. HAEs were most frequent during early treatment and declined over time. No patient with acute systemic hypersensitivity events tested positive for pegvaliase-specific IgE near the time of the event. Laboratory evidence was consistent with immune complex-mediated type III hypersensitivity. No evidence of pegvaliase-associated IC-mediated end organ damage was noted. INTERPRETATION:Despite a universal ADA response post-pegvaliase administration, adult patients with PKU achieved substantial and sustained blood Phe reductions with a manageable safety profile. FUND: BioMarin Pharmaceutical Inc.
Project description: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.
Project description:Objective:To review the pharmacology, pharmacokinetics, efficacy, safety, and place in therapy of a phenylalanine-metabolizing enzyme indicated to reduce blood phenylalanine concentrations, pegvaliase injection. Data Sources:Searches of MEDLINE (1946-September 1, 2018) were conducted using the terms pegvaliase and phenylalanine ammonia lyase (PAL). Additional data were obtained from the prescribing information, the product dossier obtained from the manufacturer, and Clinicaltrials.gov. Study Selection and Data Extraction:All English language articles related to pharmacology, pharmacokinetics, efficacy, or safety of the combination therapy in human subjects were reviewed. Data Synthesis:Pegvaliase is a pegylated PAL enzyme that converts phenylalanine to ammonia and trans-cinnamic acid. Blood phenylalanine levels were reduced by approximately 50% to 70% in patients receiving therapeutic doses of pegvaliase. However, most patients experienced adverse events. Conclusions and Relevance:The mainstay of therapy in phenylketonuria (PKU) has historically consisted of dietary restriction of phenylalanine. Pegvaliase injection is the first Food and Drug Administration (FDA)-approved enzyme substitution therapy for patients with PKU. The therapy may be a viable option for patients with documented blood phenylalanine >600?µmol/L who have failed existing management strategies.
Project description: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.
Project description:BACKGROUND: Phenylalanine ammonia lyase (PAL) is a key enzyme of the phenylpropanoid pathway that catalyzes the deamination of phenylalanine to trans-cinnamic acid, a precursor for the lignin and flavonoid biosynthetic pathways. To date, PAL genes have been less extensively studied in gymnosperms than in angiosperms. Our interest in PAL genes stems from their potential role in the defense responses of Pinus taeda, especially with respect to lignification and production of low molecular weight phenolic compounds under various biotic and abiotic stimuli. In contrast to all angiosperms for which reference genome sequences are available, P. taeda has previously been characterized as having only a single PAL gene. Our objective was to re-evaluate this finding, assess the evolutionary history of PAL genes across major angiosperm and gymnosperm lineages, and characterize PAL gene expression patterns in Pinus taeda. METHODS: We compiled a large set of PAL genes from the largest transcript dataset available for P. taeda and other conifers. The transcript assemblies for P. taeda were validated through sequencing of PCR products amplified using gene-specific primers based on the putative PAL gene assemblies. Verified PAL gene sequences were aligned and a gene tree was estimated. The resulting gene tree was reconciled with a known species tree and the time points for gene duplication events were inferred relative to the divergence of major plant lineages. RESULTS: In contrast to angiosperms, gymnosperms have retained a diverse set of PAL genes distributed among three major clades that arose from gene duplication events predating the divergence of these two seed plant lineages. Whereas multiple PAL genes have been identified in sequenced angiosperm genomes, all characterized angiosperm PAL genes form a single clade in the gene PAL tree, suggesting they are derived from a single gene in an ancestral angiosperm genome. The five distinct PAL genes detected and verified in P. taeda were derived from a combination of duplication events predating and postdating the divergence of angiosperms and gymnosperms. CONCLUSIONS: Gymnosperms have a more phylogenetically diverse set of PAL genes than angiosperms. This inference has contrasting implications for the evolution of PAL gene function in gymnosperms and angiosperms.
Project description:Phenylketonuria (PKU), a Mendelian autosomal recessive phenotype (OMIM 261600), is an inborn error of metabolism causing impaired postnatal cognitive development in the absence of treatment. We used the Pah(enu2/enu2) PKU mouse model to study oral enzyme substitution therapy with various chemically modified formulations of phenylalanine ammonia lyase (Av-p.C503S/p.C565S/p.F18A PAL). In vivo studies with the most therapeutically effective formulation (5kDa PEG-Av-p.C503S/p.C565S/p.F18A PAL) revealed that this conjugate, given orally, yielded statistically significant (p=0.0029) and therapeutically relevant reduction (~40%) in plasma phenylalanine (Phe) levels. Phe reduction occurred in a dose- and loading-dependent manner; sustained clinically and statistically significant reduction of plasma Phe levels was observed with treatment ranging between 0.3 IU and 9 IU and with more frequent and smaller dosings. Oral PAL therapy could potentially serve as an adjunct therapy, perhaps with dietary treatment, and will work independently of phenylalanine hydroxylase (PAH), correcting such forms of hyperphenylalaninemias regardless of the PAH mutations carried by the patient.